JP7005551B2 - Treatment and diagnosis of melanoma - Google Patents

Description

Cross-reference of related applications

This application claims the priority of US Provisional Patent Application No. 61 / 682,339 (filed August 13, 2012) and US Provisional Patent Application No. 61 / 784,057 (filed March 14, 2013). .. The contents of these applications are incorporated herein by reference in their entirety.

The present invention relates to the diagnosis and treatment of migratory cancer and melanoma.

Melanoma is a malignant tumor that develops from abnormal melanocytes in the lower epidermis and can metastasize to distal parts of the body via the blood and lymphatic systems. Although melanoma accounts for less than 5% of skin cancer cases, it is far more dangerous and is involved in the majority of deaths associated with skin cancer. Around the world, the incidence of melanoma continues to increase at an alarming rate, with a lifetime risk of developing melanoma as high as 1/58 for men in the United States (Non-Patent Document 1). Mortality from malignant melanoma also continues to rise dramatically around the world. According to the 2006 WHO report, about 48,000 melanoma-related deaths occur worldwide per year (Non-Patent Document 2). In the United States, it has been estimated that nearly 70,000 people will be diagnosed with melanoma during 2010 and approximately 9,000 will be expected to die from the disease (Non-Patent Document 3). ..

Although some traditional cancer therapies have been used to treat metastatic melanoma, they are not effective. Therefore, metastatic melanoma remains one of the most difficult cancers to treat and one of the most feared neoplasms. Therefore, there is a need for new drugs and methods for the diagnosis and treatment of melanoma.

Jemal et al., 2008, CA: Cancer J. et al. Clin. , 58: 71-96 Lucas et al. (2006), Environmental Burden of Disease Series. 13. World Health Organization. ISBN92-4-159440-3 American Cancer Society; www. cancer. org

The present invention addresses the above needs by providing agents and methods for the diagnosis and treatment of melanoma. The invention is based, at least in part, on the unexpected discovery of a collaborative miRNA-protein network that is deregulated in metastatic melanoma. This network contains many metastasis-suppressing and metastasis-promoting factors.

In one aspect, the invention is a method for treating a cancer, comprising administering an LXR agonist to a subject in need thereof, wherein the LXR agonist slows the spread of metastasis of the cancer. It is characterized by a method of administration in an amount sufficient to increase the expression level or activity level of ApoE to a sufficient level.

In another aspect, the invention comprises a method for treating a cancer comprising administering to a subject in need thereof an ApoE polypeptide in an amount sufficient to treat the cancer. It is a feature.

In another aspect, the invention is characterized in a method of delaying the transmission of migratory cancer, comprising administering an LXR agonist or ApoE polypeptide to a subject in need thereof.

In some embodiments of any of the methods described above, the LXR agonist is an LXRβ agonist. In certain embodiments, LXR agonists increase ApoE expression levels by at least 2.5-fold in vitro. In certain embodiments, the LXRβ agonist is more selective for LXRβ than for LXRα. In another embodiment, the LXRβ agonist has an activity on LXRβ that is at least 2.5 times greater than the activity of the agonist on LXRα. In some embodiments, the LXRβ agonist has an activity on LXRβ that is at least 10-fold greater than the activity of the agonist on LXRα. In a further embodiment, the LXRβ agonist has an activity on LXRβ that is at least 100-fold greater than the activity of the agonist on LXRα. In certain embodiments, the LXR agonist has activity on LXRβ that is at least 2.5 times the activity of the agonist on LXRα.

In some embodiments, the migratory cancer is a metastatic cancer. Metastatic cancers can include cells that exhibit migration and / or infiltration of migratory cells, and / or cells that exhibit endothelial recruitment and / or angiogenesis. In another embodiment, the migratory cancer is a cell migratory cancer. In yet still other embodiments, the cell migration cancer is a non-metastatic cell migration cancer.

The migrating cancer can be a cancer that propagates through dissemination on the surface of the peritoneal, pleural, pericardial or subarachnoid spaces. Alternatively, the migratory cancer can be a cancer that propagates through the lymphatic system or a cancer that propagates through the bloodstream.

In certain embodiments, the migratory cancer is a non-metastatic cell migratory cancer, such as ovarian cancer, mesothelioma or primary lung cancer.

In a related aspect, the invention provides a method for inhibiting or ameliorating cancer metastasis, comprising administering an LXR agonist or ApoE polypeptide.

In another aspect, the invention is a method for inhibiting the growth or growth of cancer stem cells or cancer progenitor cells, wherein the cells are LXR agonists in an amount sufficient to inhibit the growth or growth of the cells. Alternatively, a method comprising contacting with an ApoE polypeptide is provided.

In yet another aspect, the invention is a method of reducing the rate of tumor dissemination of cancer, in which a subject in need thereof is administered an LXR agonist or ApoE polypeptide in an amount sufficient to reduce the rate of tumor dissemination. Provide methods that include doing.

In yet further aspects, the invention is a method of reducing or treating metastatic nodule formation in cancer, sufficient to treat the subject in need thereof with said metastatic nodule formation in cancer. Provided are methods comprising administering a LXR agonist or ApoE polypeptide in a large amount.

In other embodiments, the cancer is breast cancer, colon cancer, renal cell cancer, non-small cell lung cancer, hepatocellular cancer, gastric cancer, ovarian cancer, pancreatic cancer, esophageal cancer, prostate cancer, sarcoma or melanoma. In some embodiments, the cancer is melanoma. In another embodiment, the cancer is breast cancer. In certain embodiments, the cancer is renal cell carcinoma. In a further embodiment, the cancer is pancreatic cancer. In another embodiment, the cancer is non-small cell lung cancer. In some embodiments, the cancer is colon cancer. In a further embodiment, the cancer is ovarian cancer.

In other embodiments, the cancer is a drug resistant cancer. In a further embodiment, the cancer is resistant to vemurafenib, dacarbazine, CTLA4 inhibitors, PD1 inhibitors or PDL1 inhibitors.

In some embodiments, the method is selected from a list consisting of any one compound of formulas I-IV or any of the compounds of compound numbers 1-39 or pharmaceutically acceptable salts thereof. Includes administration of the LXR agonist to be administered. In some embodiments, the LXR agonist is compound 1 or a pharmaceutically acceptable salt thereof. In other embodiments, the LXR agonist is compound 2 or a pharmaceutically acceptable salt thereof. In certain embodiments, the LXR agonist is compound 3 or a pharmaceutically acceptable salt thereof. In a further embodiment, the LXR agonist is compound 12 or a pharmaceutically acceptable salt thereof. In some embodiments, the LXR agonist is compound 25 or a pharmaceutically acceptable salt thereof. In other embodiments, the LXR agonist is compound 38 or a pharmaceutically acceptable salt thereof. In a further embodiment, the LXR agonist is compound 39 or a pharmaceutically acceptable salt thereof.

The method can further comprise administering an antiproliferative agent, in which case the LXR agonist and the antiproliferative agent are administered together in an amount sufficient to slow the progression of the migratory cancer. Will be done. For example, an antiproliferative agent and an LXR agonist in an amount effective for treating the subject together, respectively, within 28 days (eg, within 21 days, within 14 days, within 10 days, 7 days, respectively). Within 5 days, 4 days, 3 days, 2 days, or 1 day, or within another 24 hours (eg, 12 hours, 6 hours, 3 hours, 2 hours, or It can be administered within 1 hour or simultaneously).

In some embodiments, the method comprises administering an ApoE polypeptide. The ApoE polypeptide fragment can increase the activity or expression level of LRP1 or LRP8, and / or the ApoE polypeptide can bind to LRP1 or LRP8, and the ApoE polypeptide is the receptor binding region of ApoE ( RBR) is possible. The method can further comprise administering an antiproliferative agent, in which case the ApoE polypeptide and the antiproliferative agent together are sufficient to slow the progression of migratory cancer. Be administered. For example, an antiproliferative agent and an ApoE polypeptide in an amount effective for treating the subject together, respectively, within 28 days (eg, within 21 days, within 14 days, within 10 days, 7), respectively. Within days, within 5 days, within 4 days, within 3 days, within 2 days or within 1 day, or within another 24 hours (eg, within 12 hours, within 6 hours, within 3 hours, within 2 hours) Alternatively, it can be administered within 1 hour or simultaneously).

In some embodiments, the pharmaceutical composition may further comprise additional compounds having antiproliferative activity. Further compounds with antiproliferative activity can be found in various groups of compounds, such as chemotherapeutic agents and cytotoxic agents, differentiation inducers (eg, retinoic acid, vitamin D, cytokines), hormonal agents, immunological agents and anti-proliferative agents. It can be selected from angiogenesis agents and the like. Chemotherapeutic agents and cytotoxic agents include alkylating agents, cytotoxic antibiotics, antimetabolites, vinca alkaloids, etoposides and others (eg, paclitaxel, taxol, docetaxel, taxotere, cis-platin). Not limited to these. A list of additional compounds with antiproliferative activity can be found in L. et al. Brunton, B.I. Chamber and B. Found in Knollman (eds.), Goodman and Gilman's The Pharmaceutical Bases of Therapeutics, 12th Edition, 2011, McGraw Hill Education, New York, NY.

The above method further comprises an alkylating agent, a platinum agent, a metabolic antagonist, a topoisomerase inhibitor, an antitumor antibiotic, a thread division inhibitor, an aromatase inhibitor, a thymidyllate synthase inhibitor, a DNA antagonist, a farnesyl transferase inhibitor, and a pump. Inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNFalpha agonists / antagonists, endoserin A receptor antagonists, retinoic acid receptor agonists, immunomodulators, hormonal agents and antihormonal agents, Photodynamic agents, tyrosine kinase inhibitors, antisense compounds, corticosteroids, HSP90 inhibitors, proteosome inhibitors (eg NPI-0052), CD40 inhibitors, anti-CSI antibodies, FGFR3 inhibitors, VEGF inhibitors, MEK inhibitor, cyclin D1 inhibitor, NF-κB inhibitor, anthracyclins, histon deacetylase, kinesin inhibitor, phosphatase inhibitor, COX2 inhibitor, mTOR inhibitor, calcinulin antagonist, IMiD, or proliferative disorder May include administration of an antiproliferative compound selected from the group consisting of other agents used to treat the disease. Examples of such compounds are provided in Table 1.

In another aspect, the invention features a method for treating melanoma (eg, metastatic melanoma) in a subject in need of the treatment. The method comprises (a) a group consisting of DNAJA4, apolipoprotein E (ApoE), LRP1, LRP8, liver X receptors (LXR, eg, both LXR-alpha and LXR-beta) and miR-7 in the subject. To increase the expression or activity level of the metastatic inhibitor selected from, or (b) selected from the group consisting of miR-199a-3p, miR-199a-5p, miR-1908 and CTGF in the subject. Includes reducing the expression or activity levels of metastatic factors.

In this method, the augmenting step can be performed by administering to the subject one or more of the following (i)-(iv): (i) DNAJA4, ApoE or ApoE fragment, LRP1, LRP1, LRP8, or a polypeptide having a sequence of LXR, (ii) a nucleic acid having a sequence encoding DNAJA4, ApoE, LRP1, LRP8 or LXR, (iii) a ligand for LRP1, LRP8 or LXR, and (iv) miR-. RNAi agent encoding 7. Examples of LRP1 or LRP8 ligands include receptor binding moieties of ApoE, anti-LRP1 or anti-LRP8 antibodies, and small molecule ligands. In one example, increasing the expression level of ApoE can be done by increasing the activity level or expression level of LXR. Increasing the expression level of DNAJA4 can also be done by increasing the activity level or expression level of LXR. The activity level of LXR can be increased by administering to the subject a ligand of LXR, such as, for example, compounds of formulas I-IV as disclosed below. The step of increasing can also be performed by reducing the expression or activity level of microRNA selected from the group consisting of miR-199a-3p, miR-199a-5p and miR-1908. To this end, many techniques known in the art can be used, including the miR-Zip technique, Locked Nucleic Acid (LNA), as described in the Examples below. Techniques and antagomir techniques include, but are not limited to.

In another aspect, the invention provides a method for determining whether a subject has or is at risk of having metastatic melanoma. This method obtains a sample from a subject; in the sample, the first expression of a metastasis-promoting factor selected from the group consisting of (i) miR-199a-3p, miR-199a-5p, miR-1908 and CTGF. Levels, or (ii) to measure the second expression level of the metastatic inhibitor selected from the group consisting of DNAJA4, ApoE, LRP1, LRP8, LXR and miR-7; and the first expression level. Includes comparing one with a predetermined reference value, or comparing a second expression level with a second predetermined reference value. The subject is (a) if the first expression level is above the first predetermined reference value, or (b) if the second expression level is below the second predetermined reference value. , Is determined to have metastatic melanoma, or is at risk of having metastatic melanoma. A first predetermined reference value and a second predetermined reference value can be obtained from a control subject having no metastatic melanoma. In one embodiment, the step of measuring comprises measuring both a first expression level and a second expression level. The sample can be a body fluid sample, a tumor sample, a nevus sample or a human skin sample.

In another aspect, the invention is a transfer-promoting factor selected from the group consisting of a support having multiple unique positions and (i) miR-199a-3p, miR-199a-5p, miR-1908 and CTGF. At least one nucleic acid having a sequence complementary to the nucleic acid encoding or its complement, or (ii) transfer inhibition selected from the group consisting of DNAJA4, ApoE, LRP1, LRP8, LXR and miR-7. An array is provided that includes any combination of at least one nucleic acid having a sequence that is complementary to the nucleic acid encoding the factor or its complement. Preferably, each nucleic acid is immobilized at a unique position on the support. This array can be used for the diagnosis and prognosis of metastatic melanoma.

Therefore, the present invention also provides a kit for diagnosing the metastatic potential of melanoma in a subject. The kit is a first reagent that specifically binds to an expression product of a translocation inhibitor gene selected from the group consisting of DNAJA4, ApoE, LRP1, LRP8, LXR and miR-7, or miR-199a-3p, miR. Includes a second reagent that specifically binds to the expression product of the metastatic factor gene selected from the group consisting of -199a-5p, miR-1908 and CTGF. The second agent can be a probe having a sequence that is complementary to the suppressor gene or the promoter gene or its complement. The kit can further include reagents for performing immunoassays, hybridization assays or PCR assays. In one embodiment, the kit contained the array described above.

In another aspect, the invention provides a method of identifying compounds useful for treating melanoma or for inhibiting endothelial recruitment, cell infiltration or metastatic angiogenesis. This method comprises (i) a reporter gene encoded by a nucleic acid operably linked to the promoter of a marker gene selected from the group consisting of miR-199a-3p, miR-199a-5p, miR-1908 and CTGF. Obtaining test cells to express; (ii) exposing the test cells to the test compound; (iii) measuring the expression level of the reporter gene in the test cells; (iv) comparing the expression level to the control level; (V) If comparisons show that expression levels are below control levels, test compounds can be used to treat melanoma or to inhibit endothelial recruitment, cancer cell infiltration or metastatic angiogenesis. Includes selection as a useful candidate.

The present invention provides another method of identifying compounds useful for treating melanoma or for inhibiting endothelial recruitment, cell infiltration or metastatic angiogenesis. This method is a test cell expressing a reporter gene encoded by a nucleic acid operably linked to the promoter of a marker gene selected from the group consisting of (i) DNAJA4, ApoE, LRP1, LRP8, LXR and miR-7. To obtain; (ii) expose the test cells to the test compound; (iii) measure the expression level of the reporter gene in the test cells; (iv) compare the expression level to the control level; and (v) comparison. If the expression level is shown to be greater than the control level, the test compound is a useful candidate for treating melanoma or for inhibiting endothelial recruitment, cancer cell infiltration or metastatic angiogenesis. Includes selecting as.

In the specific method described above, the reporter gene is a standard reporter gene known in the art (eg, LaxZ gene, GFP gene or luciferase gene, etc.), or the above-mentioned metastasis-suppressing factor gene or metastasis-promoting factor gene. It is possible to be one of. In these methods, control levels can be obtained from control cells that are the same as the test cells except that they have not been exposed to the test compound.

In another aspect, the invention is a method for inhibiting endothelial recruitment in a subject in need thereof, or a method for inhibiting tumor cell infiltration in a subject in need thereof, or. A method for treating a metastatic cancer in a subject in need of the treatment, provided which the subject is administered with a drug that inhibits the expression or activity of CTGF. Subjects can be those with disorders characterized by pathological angiogenesis, including, but not limited to, cancer (eg, metastatic melanoma), eye disorders and inflammatory disorders. An example of tumor cells is metastatic melanoma cells. Examples of agents include antibodies, nucleic acids, polypeptides and small molecule compounds. In a preferred embodiment, the antibody is a monoclonal antibody.

In another aspect, the invention is a method for inhibiting endothelial recruitment in a subject in need thereof, or a method for inhibiting tumor cell infiltration in a subject in need thereof, or. A method for treating a metastatic cancer in a subject in need thereof is provided by administering to the subject an agent that increases the expression or activity of miR-7. An example of tumor cells is metastatic melanoma cells. Examples of agents include antibodies, nucleic acids, polypeptides or small molecule compounds. In one example, the agent has miR-7 activity. The nucleic acid can be an oligonucleotide. Oligonucleotides can include sequences selected from the group consisting of SEQ ID NOs: 36-38.

As used herein, "migratory cancer" refers to a cancer in which the cancer cells that form the tumor migrate and then grow as a malignant transplant at a site other than the site of the original tumor. show. Cancer cells infiltrate the lymphatic system by infiltration of lymph cells, as well as local lymph, through dissemination on the surface of the peritoneal, thoracic, pericardial or submucosal cavities to propagate into the body cavity. It migrates through transport to the nodes and distal lymph nodes, then to other parts of the body, through hematogenous transmission by infiltration of blood cells, or through infiltration into surrounding tissues. Migratory cancers include metastatic tumors and cell migration cancers, each of which is characterized by cell migration, such as ovarian cancer, mesothelioma and primary lung cancer.

As used herein, "slowing the spread of migratory cancer" means weakening or stopping the formation of new lesions, or reducing, stopping, or retrograde tumor loading. Show to let.

As used herein, a "metastatic tumor" is one in the body of a subject where the cancer cells forming the tumor metastasize through the lymphatic system or through hematogenous transmission. It has a great ability to propagate from one location to another (s), or metastasizes via the lymphatic system or through hematogenous transmission, or one location in the subject's body. Indicates a tumor or cancer that begins to spread from one place (s) to another location (s) and produces, for example, a secondary tumor in the body of the subject. Such metastatic behavior may indicate a malignant tumor. In some cases, metastatic behavior may be accompanied by an increase in tumor cell migration and / or cell infiltration behavior.

As used herein, "slowing the spread of metastases" means weakening or stopping the formation of new lesions, or reducing, stopping, or reversing tumor loading. Is shown.

The term "cancer" refers to any cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, cancers, sarcomas, leukemias and lymphomas.

As used herein, "drug-resistant cancer" refers to any cancer that is resistant to the antiproliferative agents in Table 2.

Examples of cancers that can be defined as metastatic include non-small cell lung cancer, breast cancer, ovarian cancer, colonic rectal cancer, biliary tract cancer, bladder cancer, brain cancer (glioblastoma and medullary carcinoma). ), Cervical cancer, chorionic villus cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological neoplasm, multiple myeloma, leukemia, intraepithelial neoplasia, liver cancer, lymphoma, neuroblastoma, oral cancer , Pancreatic cancer, prostate cancer, sarcoma, skin cancer (including melanoma, basal cell cancer, squamous epithelial cancer), testis cancer, interstitial tumor, embryonic cell tumor, thyroid cancer and kidney cancer, but limited to these Not done.

"Proliferation", as used in this application, involves replication or increase in similar morphology (cells) resulting from the formation of (cell) elements.

"Cell migration," as used in this application, involves infiltration of the surrounding tissue by the cancer cells and traversal of the vessel wall to exit the vasculature in the distal organs of the cancer cells.

By "cell migration cancer" is meant cancer that migrates by infiltration of the surrounding tissue by the cancer cells and across the walls of blood vessels to exit the vasculature in the distal organs of the cancer cells.

"Non-metastatic cell migration cancer" as used herein refers to a cancer that does not migrate via the lymphatic system or via hematogenous transmission.

As used herein, "cell-cell adhesion" refers to adhesion between at least two cells via interaction between a selectin molecule and a selectin-specific ligand. Cell-cell adhesion involves cell migration.

"Cell adhesion-related disorders" are defined herein as any disease or disorder that results from cell-cell adhesion or migration, or is associated with cell-cell adhesion or migration. Cell adhesion disorders also include any disease or disorder resulting from improper activation of the immune or inflammatory system, abnormal or abnormal activation. Such diseases include, but are not limited to, myocardial infarction, bacterial or viral infections, metastatic conditions (eg, cancer). The invention further comprises a method for treating cell adhesion disorders by administering an LXR agonist or ApoE polypeptide.

As used herein, "cancer stem cells" or "cancer primordial cells" are cancer cells with characteristics associated with normal stem cells, specifically all cell types found in a particular cancer sample. Shows cancer cells capable of producing. Therefore, cancer stem cells are tumorigenic, i.e., tumorigenic, perhaps in contrast to other non-tumorogenic cancer cells. Cancer stem cells can persist in tumors as a different population and can cause cancer recurrence and metastasis by giving rise to new tumors.

As used herein, "tumor dissemination" refers to the outflow of tumor cell clusters and their subsequent growth as malignant transplants at sites different from the site of the original tumor.

As used herein, a "metastatic nodule" refers to an aggregate of tumor cells in the body at a site different from the site of the original tumor.

Details of one or more embodiments of the invention are set forth in the description below. Other features, objectives and advantages of the invention will be apparent from the description and from the claims.

Systematic identification of miR-1908, miR-199a-3p and miR-199a-5p as endogenous promoters of human melanoma metastasis. (A) A heat map illustrating dispersion-normalized microarray expression values of miRNAs upregulated for their respective parent cells in independent MeWo-transferred and A375-transferred derivatives. The color map shows the change in standard deviation from the mean of each heatmap column. (B) MiRNAs found to be upregulated by microarray hybridization were confirmed by qRT-PCR in the MeWo-LM2 transposable derivative. n = 3. (C) Bioluminescence of metastatic colony formation in the lung after intravenous infusion of 4 × 10 ⁴ parental MeWo cells overexpressing precursors for miR-199a, miR-1908, miR-214 or control hairpins. Sense imaging plot. The lungs were removed 63 days after injection and stained with H & E. n = 5. (D) Expresses a short hairpin (miR-Zip) or control sequence (shCTRL) that inhibits miR-1908 (m1908 KD), miR-199a-3p (m199a3p KD), miR-199a-5p (m199a5p KD) 4 × 10 Bioluminance imaging plots and H & E stained lungs corresponding to lung metastases after intravenous infusion of ⁴ LM2 cells. Lungs were removed 49 days after injection and stained with H & E. n = 5-8. (E) Colonization in the lung by 2 × 10 ⁵ A375-LM3 metastatic derivatives with miR--1908, miR-199a-3p, miR-199a-5p or miR-Zip-induced silencing of the control sequence. It was quantified on day 42 by bioluminescence imaging. n = 5-8. (F) Expression levels of miR-199a-3p, miR-199a-5p and miR-1908 of non-metastatic primary melanoma skin lesions (n = 38) and metastatic primary melanoma skin lesions (n = 38) obtained from MSKCC patients. It was determined in a blinded manner by qRT-PCR in the cohort of n = 33). n = 71. All data are expressed as mean ± SEM. ^* P <0.05, ^** p <0.01, ^*** p <0.001. See also Figure 12. miR-1908, miR-199a-3p and miR-199a-5p exhibit a dual cell-autonomous / cell-non-autonomous role in regulating melanoma metastasis progression. (A) 1 × 10 ⁶ parental MeWo cells overexpressing miR-199a, miR-1908 or control hairpins were subcutaneously injected into immunodeficient mice and the primary tumor volume was monitored over time. n = 4-6. (B) 1 × 10 ⁵ parental MeWo cells overexpressing miR-199a, miR-1908 or control hairpins were infiltrated through Transwell's Matrigel-coated inserts for 24 hours and the base of each insert. The number of cells infiltrating the side was quantified. n = 7. (C to D) 1 × 10 ⁵ highly metastatic MeWo-LM2 cells (C) and A375 with miR-199a-3p, miR-199a-5p, miR-1908 or control sequence miR-Zip induction inhibition -LM3 cells (D) were subjected to a cell infiltration assay. n = 6-8. (E) 5 × 10 ⁴ MeWo cells overexpressing miR-199a, miR-1908 or control hairpins were seeded at the bottom of the well and 1 × 10 ⁵ human umbilical vein endothelial cells (HUVEC) were translocated. They were allowed to migrate towards cancer cells for 16 hours through the well inserts. Endothelial mobilization capacity was measured by quantifying the number of HUVECs migrating to the base side of each insert. n = 7. (FG) Endothelium by 5 × 10 ⁴ MeWo-LM2 cells (F) and A375-LM3 cells (G) inhibited for miR-199a-3p, miR-199a-5p, miR-1908 or control sequences mobilization. n = 6-10. (H) Metastatic formed after intravenous infusion of 2 × 10 ⁵ highly metastatic MeWo-LM2 cells depleted for miR-199-3p, miR-199a-5p, miR-1908 or control sequences. Cumulative percentage plot of percentage vascular density distribution for nodules. Lung sections were immunohistochemically double-stained for human vimentin (blue) and MECA-32 (red) to determine the percentage MECA-32 positive area within each metastatic nodule distinguished based on vimentin staining. Quantified. n = 211 nodules (control KD); n = 60 nodules (m199a3p KD); n = 138 nodules (m199a5p KD); n = 39 nodules (m1908 KD). All data are expressed as mean ± SEM. Scale bar, 100 μm. See also FIG. Identification of ApoE and DNAJA4 as common target genes for miR-199a and miR-1908. (A) ApoE and DNAJA4 mRNA levels measured by qRT-PCR in low metastatic MeWo cells overexpressing miR-199a, miR-1908 or control hairpins and high metastatic MeWo-LM2 cells. Heat map to show. The color map illustrates the change in standard deviation from the average of each heatmap column. (B) Wild-type ApoE 3'UTR / CDS luciferase fusion and DNAJA4 3'UTR / CDS luciferase fusion, or miRNA target site in parental MeWo cells that overexpress miR-199a, miR-1908 or control hairpins. A heterologous luciferase reporter assay that measures the stability of mutant ApoE 3'UTR / CDS luciferase fusions and DNAJA4 3'UTR / CDS luciferase fusions. n = 3-4. (C) Wild-type ApoE 3'UTR / CDS luciferase fusion and DNAJA4 3'UTR in MeWo-LM2 cells in which expression of miR-199a-3p, miR-199a-5p, miR-1908 or control sequence was silenced. / Stability of CDS luciferase fusion. n = 4. (D) Schematic of experimentally derived models of ApoE 3'UTR / CDS targeting and DNAJA4 3'UTR / CDS targeting with miR-199a-3p, miR-199a-5p and miR-1908. (E) Wild-type and miRNA target site mutant ApoE 3'UTR / CDS luciferase fusions and DNAJA4 3'UTR / CDS luciferase fusions in highly metastatic MeWo-LM2 derivatives and their low metastatic parent cell lines. Luciferase activity of the thing. n = 4. (F) Matrigel infiltration ability by 1 × 10 ⁵ MeWo-LM2 cells expressing the control vector or overexpressing ApoE or DNAJA4. n = 4. (G) Endothelial mobilization ability by 5 × 10 ⁴ MeWo-LM2 cells transduced with a control vector or an overexpression vector for ApoE or DNAJA4. n = 6. (HI) Low-metastatic parental MeWo cells transduced by short lentiviral hairpins targeting ApoE, DNAJA4 or control sequences are evaluated for their Matrigel infiltration capacity (H) and endothelial cell recruitment capacity (I). did. n = 6-8. All data are expressed as mean ± SEM. Scale bar, 100 μm. See also FIG. Direct targeting of ApoE and DNAJA4 with miR-199a and miR-1908 promotes metastatic infiltration, endothelial recruitment and colonization. (A-D) Control hairpins, or shRNAs that target ApoE or DNAJA4 in miR-1908 inhibition status (m1908 KD; A, B) or miR-199a-5p inhibition status (m199a5p KD; C, D). Highly metastatic LM2 cells expressing the above were subjected to cell infiltration assay (A, C) and endothelial mobilization assay (B, D). n = 6-8. (EF) Control hairpins, or 1x expressing hairpins that target ApoE, DNAJA4 or control sequences in miR-1908 silencing status (E) or miR-199a-5a silencing status (F). Bioluminescence imaging plots and H & E stained lungs representing lung metastases after intravenous infusion of 105 ^LM2 cells. n = 5. Parental MeWo cells overexpressing (GH) ApoE or DNAJA4 or expressing the control vector in the context of miR-1908 overexpression were analyzed for Matrigel infiltration phenotype (G) and endothelial recruitment phenotype (H). .. (IJ) Control shRNA, or A375-LM3 derivative expressing shRNA that targets ApoE and DNAJA4, is a cocktail of LNA that targets miR-199a-3p, miR-199a-5p, and miR-1908. , Or was transfected with a control LNA and analyzed in the Matrigel infiltration assay (I) and the endothelial mobilization assay (J). n = 4. (K) Blood vessels represented by a cumulative percentage plot for metastatic nodules formed by MeWo-LM2 cells that are inhibited for miR-1908 and transduced by shRNA that targets ApoE, DNAJA4 or control sequences. Density distribution. Lung sections from FIG. 4E were immunocytochemically double stained with human vimentin (blue) and the endothelial marker MECA-32 (red). Percentage MECA-32 positive areas within each vimentin-positive nodule were quantified. n = 39 nodules (shCTRL); n = 97 (shAPOE ¹ ); n = 38 (shAPOE ² ); n = 200 (shDNAJA4 ¹ ); n = 19 ( ^shDNAJA42 ). All data are expressed as mean ± SEM. Scale bar, 100 μm. See also FIG. ApoE secreted by melanoma cells inhibits melanoma infiltration and endothelial recruitment, while genetic deletion of ApoE accelerates metastasis. (A-B) in conditioned medium obtained from MeWo-LM2 transposable derivatives and their parent cells (A) and LM2 cells (B) silenced for miR-199a-5p, miR-1908 or control sequences. Extracellular ApoE levels quantified by ELISA. n = 3. (C) ApoE neutralizing antibody 1D7 (10-40 μg / mL) or IgG (40 μg / mL) was added to the cell medium and infiltration of Matrigel by parental MeWo cells was evaluated. n = 4-6. (D) Endothelial recruitment by parental MeWo cells in the presence of 1D7 antibody (40 μg / mL) or control IgG antibody (40 μg / mL). n = 4. (E) The phenotype of Matrigel infiltration and endothelial recruitment was evaluated in LM2 cells in the presence of bovine serum albumin (BSA) (100 μM) or recombinant ApoE3 (100 μM) added to the cell medium. n = 7-10. (FG) LM2 cells with silenced expression of miR-199a-3p, miR-199a-5p, miR-1908 or control sequence in the presence of IgG antibody or ApoE neutralized 1D7 antibody (40 μg / mL). , Matrigel infiltration ability (F) and endothelial mobilization ability (G) were investigated. n = 5-6. (H) ApoE levels quantified by ELISA in conditioned medium obtained from parental MeWo cells transduced with DNAJA4 or shRNA targeting the control sequence. n = 3. (IJ) Parental MeWo cells with shRNA-induced silencing of DNAJA4 in the presence of either BSA (100 μM) or recombinant ApoE3 (100 μM) in the presence of Matrigel infiltration phenotype (I) and endothelial mobilization phenotype (J). Was analyzed. n = 4. (K) Array-based ApoE expression levels in nevus sample (n = 9), primary melanoma sample (n = 6) and distal melanoma metastatic sample (n = 19). (L) Highly metastatic MeWo-LM2 cells were incubated in the presence of recombinant ApoE3 or BSA at 100 μg / mL. After 24 hours, 4 × 10 ⁴ cells were intravenously injected into NOD-SCID mice and colonization in the lung was monitored by bioluminescence imaging. n = 6. (M) Lung metastases by 5 × 10 ⁴ B16F10 mouse melanoma cells injected intravenously into ApoE genetically null C57BL / 6 mice or their wild-type control littermates. Lung bioluminescence quantification and typical H & E stained lungs correspond 19 days after infusion. n = 8-18. All data are expressed as mean ± SEM. Scale bar, 100 μm. Identification of different melanoma and endothelial cell receptors that mediate the effects of ApoE on melanoma infiltration and endothelial recruitment. (A) Matrigel infiltration capacity is transduced by siRNA targeting LDLR, VLDLR, LRP8, LRP1 or control sequences in the presence of either BSA (100 μΜ) or recombinant ApoE3 (100 μΜ) 1 × 10 ⁵ It was examined in LM2 cells. n = 4-7. (B) 1 × 10 ⁵ MeWo-LM2 cells transduced by miR-1908 or short hairpins targeting the control sequence are transfected with siRNA or control siRNA targeting LRP1 for Matrigel infiltration assay. Served. n = 4. (C) Bioluminescence imaging of colonization in the lung by 1 × 10 ⁵ LM2 cells transduced by LRP1 or siRNA targeting the control sequence in the context of miR-1908 inhibition. n = 5. (D) 1 × 10 ⁵ endothelial cells preincubated with BSA (100 μΜ) or recombinant ApoE3 (100 μΜ) for 24 hours were analyzed for endothelial mobilization phenotype by 5 × 10 ⁵ LM2 cells. n = 3-4. (E) 1 × 10 ⁵ endothelial cells are transduced with LDLR, VLDLR, LRP1, LRP8 or siRNA targeting the control sequence and transwelled towards miR-1908 or LM2 cells that are inhibited for the control sequence. I let it run in the system. n = 4-12. (F) Transwell migration by 1 × 10 ⁵ endothelial cells in the presence of IgG antibody (40 μg / mL) or 1D7 antibody (40 μg / mL) added to cell medium. n = 6-8. (G) Transwell migration by 1 × 10 ⁵ endothelial cells transduced by LRP8 or siRNA targeting the control sequence in the presence of BSA (100 μΜ) or recombinant ApoE3 (100 μΜ). n = 6-7. (H) 1 × 10 ⁵ endothelial cells were transduced with LRP8 or siRNA targeting the control sequence and transwell chemotactic migration was evaluated along the ApoE gradient. n = 6-8. (I) Ventral flank of mice containing BSA (10 μg / mL), VEGF (400 ng / mL) + BSA (10 μg / mL), or VEGF (400 ng / mL) + recombinant ApoE3 (10 μg / mL). Endothelial recruitment into a Matrigel plug implanted subcutaneously above. n = 3-6. (J) Vascular density within lung metastatic nodules formed after intravenous injection of 5 × 10 ⁴ B16F10 mouse melanoma cells into wild-type or ApoE genetic null mice. Lung sections from FIG. 5M were immunohistochemically stained for MECA-32 to quantify the percentage MECA-32 positive area within each metastatic nodule contoured based on cell pigmentation. .. n = 17-20. All data are expressed as mean ± SEM. Scale bar, 100 μm. Clinical and therapeutic cooperativity (AD) between miR-199a-3p, miR-199a-5p and miR-1908 in melanoma metastasis. MiR-199a-3p expression level (A), miR-199a-5p expression level (B), miR-1908 expression level (C), or total expression level of three miRNAs (D) in the patient's primary melanoma lesion. The Kaplan-Meier curve for the MSKCC cohort (n = 71), which represents the metastasis-free survival of patients as a function of). Patients whose primary or primary tumor miRNA expression level or total miRNA expression level (sum of expression of miR-199a-3p, miR-199a-5p and miR-1908) exceeds the median for the population are designated as positive miRNA expression (red). Patients whose primary tumors expressed these given miRNAs at lower levels than this median were classified as negative miRNA expression (blue). (E) A combination of LNAs that individually target miR-1908, miR-199a-3p or miR-199a-5p, respectively, or a combination of LNAs that target all three miRNAs, or high transfected by a control LNA. Lung metastasis by metastatic LM2 cells. Forty-eight hours after transfection, 1 × 10 ⁵ cells were intravenously injected into immunodeficient mice. n = 5-6. (F) Intracardiac to thymic nude mice by control LNA (LNA-CTRL) or LNA cocktails (LNA-3miRNAs) targeting miR-1908, miR-199a-3p, miR-199a-5p. Systemic metastasis by 1 × 10 ⁵ LM2 cells transfected 48 hours before infusion. n = 5. (G) Number of systemic metastatic lesions arising from LM2 cells of LNA-CTRL and LM2 cells of LNA-3miRNAs on the 28th day after intracardiac injection. n = 5. (HI) Bioluminescence signal quantification of bone metastases (H) and brain metastases (I) 28 days after intracardiac injection of LM2 cells of LNA-CTRL and LM2 cells of LNA-3miRNAs. n = 5. (J) 4 × 10 ⁴ highly metastatic MeWo-LM2 cells are injected tail vein into immunocompromised mice to target miR-1908, miR-199a-3p and miR-199a-5p. Treatment was performed by intravenous administration at a total dose of 12.5 mg / kg with a cocktail of in vivo optimized LNA or twice a week for 4 weeks with simulated PBS control. Colonization in the lung was evaluated by bioluminescence imaging. Representative H & E stained lungs removed on day 56 are shown. n = 5-6. (K) A model of miRNA-dependent regulation of metastatic infiltration, endothelial recruitment and colony formation in melanoma through targeting ApoE-mediated melanoma cell LRP1 receptor signaling and endothelial cell LRP8 receptor signaling. MiRNA-dependent targeting of ApoE / LRP1 signaling promotes cancer cell infiltration and endothelial recruitment through the induction of CTGF. (A) Heat map of dispersion-normalized CTGF expression levels in the following cells as determined by qRT-PCR analysis: (1) MeWo parent cells and MeWo-LM2 cells, (2) miR-199a, miR-1908 or MeWo parent cells that overexpress control hairpins, as well as (3) MeWo parent cells transduced by short hairpins that target ApoE or control sequences. The color map shows the change in standard deviation from the mean. (B) CTGF levels in conditioned medium from MeWo parent cells with ApoE knockdown as determined by ELISA. n = 6; p-values are based on one-sided Student's t-test. (C) CTGF levels quantified by ELISA in conditioned medium from highly metastatic MeWo-LM2 cells treated with recombinant ApoE in the context of LRP1 knockdown or control knockdown. n = 3-4; p-values are based on one-sided Student's t-test. Parental MeWo cells with (DE) shRNA-induced ApoE knockdown are transfected with (1) CTGF or an irrelevant siRNA targeting the control sequence, or (2) CTGF neutralizing antibody (20 μg / mL). ) Or IgG control antibody (20 μg / mL) and cells were subjected to cell infiltration assay (D) and endothelial mobilization assay (E). n = 6-8; p-values are based on one-sided Student's t-test. The scale bar indicates 100 μM. All data are expressed as mean ± SEM. CTGF mediates miRNA-dependent metastatic infiltration, endothelial recruitment and colonization. (A) Blocking antibodies (20 μg) targeting CTGF of 1 × 10 ⁵ parental MeWo cells expressing control hairpins or overexpressing miR-199a or miR-1908, as shown in the figure. It was subjected to a transwell cell infiltration assay in the presence of / mL) or control IgG antibody (20 μg / mL). n = 4-10; p-values are based on one-sided Student's t-test. All data are expressed as mean ± SEM. (B) Endothelial recruitment by parental MeWo cells expressing control hairpins or overexpressing miR-199a or miR-1908. At the start of the assay, neutralizing antibody (20 μg / mL) targeting CTGF or control IgG antibody (20 μg / mL) was added to the endothelial cells as indicated and 1 × 10 ⁵ endothelial cells were transwelled into the transwell assay. In 5 × 10 ⁴ cancer cells were migrated. n = 3-8; p-values are based on one-sided Student's t-test. (C) Bioluminescence imaging of lung metastases by 5 × 10 ⁴ parental MeWo cells knocked down for CTGF in the context of miR-199a or miR-1908 overexpression. n = 5-6; p-values were determined using the one-sided Mann-Whitney t-test. All data are expressed as mean ± SEM. Treatment with the LXR agonist GW3965 increases ApoE levels in melanoma cells and suppresses cancer cell infiltration, endothelial recruitment and metastatic colonization. (AB) Parent MeWo cells were incubated in the presence of DMSO or GW3965 at the indicated concentrations. After 48 hours, total RNA was extracted and ApoE levels (A) and DNAJA4 levels (B) were determined by qRT-PCR. n = 3. (C) Cell infiltration by 1 × 10 ⁵ parental MeWo cells pretreated with GW3965 or DMSO for 48 hours. n = 6-7; p-values are based on one-sided Student's t-test. All data are expressed as mean ± SEM. (D) Endothelial recruitment by 5 × 10 ⁴ parental MeWo cells pretreated with GW3965 or DMSO for 48 hours. n = 6-7; p-values are based on one-sided Student's t-test. (E) Mice were fed a grain-based solid feed or a control diet containing GW3965 (20 mg / kg). After 10 days, 4 × 10 ⁴ parental MeWo cells were injected into the tail vein into mice, and these mice were continuously fed a GW3965-containing solid diet or a control diet throughout the experimental period. Colonization in the lung was evaluated by bioluminescence imaging. n = 5-6; p-values were obtained using the one-sided Mann-Whitney t-test. All data are expressed as mean ± SEM. Identification of miR-7 as an endogenous inhibitor of melanoma metastasis. (A) Bio-metastatic colonization in the lung after intravenous infusion of 4 × 10 ⁴ parental MeWo cells (miR-7 KD) expressing short hairpins (miR-Zip) that inhibit miR-7. Luminescence imaging plot. The lungs were removed 63 days after injection and stained with H & E. n = 5. (B) Lung metastasis by 4 × 10 ⁴ LM2 cells overexpressing precursor or control hairpin for miR-7. Colonization in the lungs was monitored weekly by bioluminescence imaging and removed 77 days after injection of the lungs. n = 5. All data are expressed as mean ± SEM; p-values were determined using the one-sided Mann-Whitney t-test. ^* P <0.05, ^** p <0.01. In vivo selection for highly metastatic human melanoma cell line derivatives and identification of miR-199a-3p, miR-199a-5p and miR-1908 as metastatic factors miRNA. (AB) MeWo-LM2 metastatic derivative (A) and A375-LM3 metastatic derivative (B) and bioluminance imaging and H & E staining of lung metastases corresponding to their respective parent cell lines. A representative image of the lungs. 4 × 10 ⁴ MeWo-Par / MeWo-LM2 cells and 1 × 10 ⁵ A375-Par / A375-LM3 cells were intravenously injected into NOD-SCID mice and the lungs were injected on days 72 and 49. Each was taken out and stained with H & E. n = 4-5. (C) Expression levels of miR-199a-5p, miR-199a-3p, miR-1908 and miR-214 were determined by qRT-PCR in A375-LM3 metastatic derivatives and their parent cells. n = 3. (D) Parental MeWo cells are pre-miRNA hairpins that express control hairpins or give rise to miR-199a (both miR-199a-3p and miR-199a-5p), miR--1908 or miR-214. Transduced by a retrovirus expressing the construct. The expression level of the target miRNA was determined by qRT-PCR. n = 3. (E) H & E stained lung sections from FIG. 1C were analyzed for the number of metastatic nodules resulting from parental MeWo cells overexpressing miR-199a, miR-1908 or control hairpins. n = 3. (F) H & E stained lung sections from FIG. 1D showing the number of metastatic nodules formed by LM2 cells with silenced expression of miR-199a-3p, miR-199a-5p, miR-1908 or control sequences. Analyzed in. n = 3. All data are expressed as mean ± SEM. miR-199a and miR-1908 inhibit proliferation in vitro and selectively promote cell infiltration and endothelial recruitment. (A) 2.5 × 10 ⁴ MeWo cells overexpressing miR-199a, miR-1908 or control hairpins were seeded in triplets and live cells were counted after 5 days. n = 3. (B) 1 × 10 ⁵ low metastatic parental MeWo cells and high metastatic LM2 cells were compared for their ability to infiltrate through Matrigel in a transwell assay. n = 3-4. (C) 1 × 10 ⁵ endothelial cells were seeded on a 6-well plate to form a monolayer. 2 × 10 ⁵ parental MeWo cells overexpressing miR-199a, miR-1908 or control hairpins were seeded onto the endothelial monolayer and incubated for 30 minutes. Each monolayer was then imaged to quantify the number of cancer cells adhering to endothelial cells. n = 3. (D) 1 × 10 ⁶ parental MeWo cells overexpressing miR-199a, miR-1908 or control hairpins were seeded on a low adhesive plate containing a cell medium supplemented with 0.2% methylcellulose. .. After 48 hours in suspension, the number of dead and live cells was quantified. n = 3. (E) 5 × 10 ⁵ parental MeWo cells overexpressing miR-199a, miR-1908 or control hairpins were seeded in 6-well plates and incubated in low serum medium for 48 hours, after which the number of live cells was increased. Quantified. n = 4. (F) Colonization by parental MeWo cells overexpressing miR-199a, miR-1908 or control hairpins. Fifty cells were seeded on 6 cm plates and the number of colonies formed was quantified after 2 weeks. n = 4. (G) 5 × 10 ⁴ parental MeWo cells and LM2 cells were seeded at the bottom of the wells and their endothelial cell mobilization capacity was evaluated. n = 6-8. (H) Percentage vascular density shown as a cumulative percentage plot for metastatic nodules formed by parental MeWo cells overexpressing miR-199a, miR-1908 or control hairpins. Lung sections from FIG. 1C were immunohistochemically double-stained for human vimentin and MECA-32, and the MECA-32 positive area for the total nodular area provided by human vimentin staining was quantified using ImageJ. n = 43 nodules (control); n = 117 nodules (miR-199a OE); n = 55 nodules (miR-1908 OE). All data are expressed as mean ± SEM. Scale bar, 100 μm. miR-199a and miR-1908 target ApoE and DNAJA4 convergently and cooperatively. (A) Venn diagram showing an integrated experimental effort leading to the identification of presumed target genes common to miR-199a-3p, miR-199a-5p and miR-1908. Transcriptome profiling of genes that are downregulated by more than 1.5 times when each miRNA is overexpressed, and genes that are upregulated by more than 1.5 times when each miRNA is silenced. And also with genes that are downregulated by more than 1.5-fold difference in metastatic LM2 cells relative to their parent cell line. (B-D) Parent MeWo cells (B) overexpressing miR-199a, miR-1908 or control hairpins, parent MeWo cells and their highly metastatic LM2-derived cell lines (C), and miR-199a- Expression levels of ApoE and DNAJA4 as measured by qRT-PCR in MeWo-LM2 cells (D) with 3p, miR-199a-5p, miR-1908 or control sequence miR-Zip mediated silencing. n = 3. (E) Target sites of miR-199a-3p, miR-199a-5p or miR-1908 in highly metastatic LM2 cells with inhibition of miR-199a-3p, miR-199a-5p, miR-1908 or control sequence. A heterologous luciferase reporter assay that measures the stability of mutant ApoE 3'UTR / CDS luciferase fusions and DNAJA4 3'UTR / CDS luciferase fusions. n = 3-4. (F) MeWo-LM2 cells were transduced with a retrovirus expressing a control vector or a retrovirus expressing an overexpression vector producing ApoE or DNAJA4. The expression level of the target gene was determined by qRT-PCR. (G) Expression levels of ApoE and DNAJA4 as determined by qRT-PCR in parental MeWo cells transduced with lentiviral shRNA targeting ApoE, DNAJA4 or control sequences. All data are expressed as mean ± SEM. Epitasis interactions (AD) between miR-199a / miR-1908 and ApoE / DNAJA4. MeWo-LM2 cells, miR-Zip-induced silencing status (A, B) of miR-1908, miR-Zip-induced silencing status of miR-199a-5p (C, D), or control sequence miR. -In the context of Zip-induced silencing, transduction was performed with lentiviral shRNA (A, C) targeting ApoE, lentivirus shRNA (B, D) targeting DNAJA4, or control shRNA. The level of the target gene was analyzed by qRT-PCR. (E) 1 × 10 ⁵ LM2 cells expressing shRNA (independent of the shRNA used in FIG. 4E) targeting the control hairpin, or ApoE, DNAJA4 or control sequence in the context of miR-1908 inhibition. Bioluminescence imaging of lung metastases by. Representative bioluminescence images and H & E stained lungs correspond to 42 days post-injection. n = 5. (FG) Expression levels of ApoE and DNAJA4 are expressed in the context of a control vector or an overexpression vector for ApoE or DNAJA4 in the context of miR-1908 overexpression (F) or miR-199a overexpression (G). It was analyzed by qRT-PCR in parental MeWo cells transduced by the retrovirus. (HI) Parental MeWo cells that overexpress ApoE or DNAJA4 or express the control vector in the context of miR-199a overexpression were examined for infiltration phenotype (H) and endothelial mobilization phenotype (I). n = 7-8. (J) Bioluminescence imaging of lung metastases by 4 × 10 ⁴ parental MeWo cells overexpressing ApoE or DNAJA4 or expressing a control vector in the context of miR-1908 overexpression. Representative bioluminescence images and H & E stained lungs correspond 56 days after infusion. n = 4-8. (K) ApoE and DNAJA4 expression levels as determined by qRT-PCR in a highly transposable A375-LM3 derivative transduced by a lentivirus that expresses ApoE and DNAJA4 or a shRNA construct that targets a control sequence. All data are expressed as mean ± SEM. Scale bar, 100 μm. Extracellular ApoE inhibits the phenotype of melanoma infiltration and endothelial recruitment regardless of any effect on the proliferation and survival of cancer or endothelial cells. (A) Extracellular ApoE levels were measured by ELISA in conditioned medium from MeWo cells overexpressing miR-199a, miR-1908 or control hairpins. n = 3. (B-C) 3 × 10 ⁴ MeWo-LM2 cells (B) or endothelial cells (C) were cultured in the presence of BSA (100 μΜ) or APOE (100 μΜ) for cell proliferation and the number of live cells. Monitored over time by counting at each indicated time point. n = 3. (DE) Survival of MeWo-LM2 cells (D) or endothelial cells (E) in the presence of serum starvation in the presence of BSA (100 μΜ) or APOE (100 μΜ). n = 3. (FG) Parent MeWo cells (F) transduced with ApoE mRNA expression levels by a lentivirus expressing a control hairpin or a short hairpin construct targeting DNAJA4, and a control vector, or DNAJA4. Was evaluated in LM2 cells (G) transduced by a retrovirus expressing an overexpression vector for. n = 3. LM2 cells transduced with a (HI) control vector or a retrovirus expressing an overexpression vector for DNAJA4 are neutralized with IgG antibody (40 μg / mL) or 1D7 antibody (40 μg / mL) (ApoE neutralization). In a transwell assay in the presence of (antibodies), their ability to infiltrate through Matrigel (H; n = 6-8) and mobilize endothelial cells (I; n = 4) were evaluated. All data are expressed as mean ± SEM. ApoE inhibits cell infiltration and endothelial recruitment by targeting the LRP1 receptor on melanoma cells and the LRP8 receptor on endothelial cells. (A) 1 × 10 ⁵ LM2 cells transduced by siRNA against LRP1 or control sequences were analyzed for their ability to infiltrate through Matrigel. n = 9-12. (B) 1 × 10 ⁵ MeWo-LM2 cells inhibited for miR-199a-5p or control sequences were transfected with LRP1 targeted siRNA or control siRNA and their matrigel infiltration capacity was investigated. n = 4. (C) Representative H & E stained lungs removed on day 56 from NOD-SCID mice injected with MeWo-LM2 miR-1908 KD cells transduced by control siRNA or LRP1 targeted siRNA (FIG. 6C). See). (DE) 5 × 10 ⁴ MeWo-LM2 cells (D; n = 8), which transfect 1 × 10 ⁵ endothelial cells with LRP8 targeted siRNA or control sequences and express short control hairpins, Alternatively, transwell migration was performed towards 5 × 10 ⁴ MeWo-LM2 cells (E; n = 4) that were inhibited for miR-199a-5p or control sequences. All data are expressed as mean ± SEM. Scale bar, 100 μm. LNA-type inhibition of miR-199a and miR-1908 suppresses melanoma metastasis. (A) In vitro cell proliferation by 2.5 × 10 ⁴ MeWo-LM2 cells transduced with a control LNA or a cocktail of LNAs targeting miR-199a-3p, miR199a-5p and miR-1908. The number of living cells was quantified after 5 days. n = 3. (B) Colonization in the lung by a control LNA or a highly metastatic A375-LM3 derivative transfected with a cocktail of LNAs targeting miR-199a-3p, miR199a-5p and miR-1908. Forty-eight hours after transfection, 5 × 10 ⁵ cells were intravenously injected into NOD-SCID mice and colonization in the lung was determined by measuring bioluminescence 35 days later. n = 5-6. (C) Weight of mice treated with LNA cocktails targeting these three miRNAs or simulated PBS control treatment (FIG. 7J) was monitored twice weekly. n = 5-6. All data are expressed as mean ± SEM. Activation of LXRβ signaling suppresses melanoma cell infiltration and endothelial recruitment. (A) A heat map showing the expression levels of LXR isotypes and RXR isotypes in the NCI-60 melanoma cell line collection based on the microarray. Heatmaps for these genes are extracted from the larger heatmaps of the nuclear hormone receptor family (FIG. 20). The colormap legend shows the change in standard deviation for the expression value of each receptor relative to the average expression value of all microarray profiled genes (over 39,000 transcription variants) in each cell line. (B) 1 × 10 ⁵ MeWo human melanoma cells, 5 × 10 ⁴ HT-144 human melanoma cells, 5 × 10 ⁵ SK-Mel-2 human melanoma cells and 5 × 10 ⁴ SK- Cell infiltration by Mel-334.2 human melanoma cells. Cells were treated with DMSO, GW3965, T0901317 or bexarotene at 1 μM for 72 hours and subjected to a Matrigel infiltration assay in transwell. n = 48. (C) 5 × 10 ⁴ MeWo human melanoma cells, HT-144 human melanoma cells, SK-Mel-2 human melanoma cells and SK-Mel-334.2 human melanoma cells, these melanoma cells at 1 μM. After treatment with DMSO, GW3965, T0901317 or bexarotene for 72 hours, transwell migration assays were tested for their ability to recruit 1 × 10 ⁵ endothelial cells. n = 4-8. (D to E) 1 × 10 ⁵ MeWo human melanoma cells (D) and 1 × 10 ⁵ HT-144 human melanoma cells (E) expressing control shRNA or an LXRα or LXRβ targeting shRNA. Was treated with DMSO, GW3965 or T0901317 at 1 μM for 72 hours before subjecting to a cell infiltration assay. n = 4-12. (FG) 5 × 10 ⁴ MeWo cells (F) and 5 × 10 ⁴ HT-144 cells (G) transduced by a lentiviral shRNA targeting LXRα or LXRβ, or control shRNA. Was treated with DMSO, GW3965 or T0901317 at 1 μM for 72 hours and tested for their ability to recruit 1 × 10 ⁵ endothelial cells in a transwell migration assay. n = 7-8. All data are expressed as mean ± SEM. Scale bar, 50 μm. ^* P <0.05; ^** p <0.01; ^*** p <0.001, ^*** p <0.0001. Analysis of nuclear hormone receptor expression in melanoma and its effect on LXR and RXR agonists on in vitro cell growth (these are associated with FIGS. 19 (AG)). (A) A heat map showing microarray-based expression levels of all nuclear hormone receptor family members across the NCI-60 collection of various melanoma strains. The expression level of each receptor is shown as a standard deviation number below or above the average expression level of all genes (> 39,000 transcription variants) detected by the microarray in each cell line. (B) 2.5 × 10 ⁴ MeWo human melanoma cells, HT-144 human melanoma cells or SK-Mel-334.2 human melanoma cells were seeded in 6-well plates and DMSO, GW3965, T0901317 or 1 μM. It was cultured in the presence of bexarotene. Live cells were counted 5 days after seeding. n = 3-6. (C) 2.5 × 10 ⁴ MeWo cells, HT-144 cells or SK-Mel-334.2 cells were placed in triplets and placed in a medium containing DMSO, GW3965, T0901317 or bexaloten at 1 μM for 5 days. After incubation, the number of dead cells was quantified using trypan blue dead cell staining. n = 3. (DG) Determined by qRT-PCR in MeWo human melanoma cells (D, E) and HT-144 human melanoma cells (F, G) expressing control shRNA or NXRα or LXRβ targeting shRNA. Relative expression of LXRα and LXRβ. All data are expressed as mean ± SEM. Therapeutic LXR activation inhibits melanoma tumor growth. (AB) Primary tumor growth by 5 × 10 ⁴ B16F10 mouse melanoma cells injected subcutaneously into C57BL / 6-WT mice. After tumor growth reaches 5 mm ^{3-10 mm 3 in} volume, mice are continuously fed with a control solid feed or a solid feed supplemented with GW3965 (20 mg / kg / day or 100 mg / kg / day) (A). ), Or a solid feed (20 mg / kg / day) (B) supplemented with T0901317. The representative tumor image shown corresponds to the tumor removed on the last day (d12). n = 10 to 18 (A), 8 to 10 (B). (C to E) 1 × 10 ⁶ MeWo human melanoma cells (C), 7.5 × 10 ⁵ SK-Mel-334.2 human melanoma cells (D) and 2 injected subcutaneously into immunocompromised mice. × 10 Primary tumor growth by ⁶ SK-Mel-2 human melanoma cells (E). After tumor growth reached 5 mm ^{3-10 mm 3 in} volume, mice were randomly assigned to a control diet or a diet supplemented with GW3965 (20 mg / kg or 100 mg / kg as shown). The tumor image shown corresponds to the last day of measurement. n = 6 to 34 (C), 8 (D), 5 (E). (F) 5 × 10 ⁴ B16F10 cells were subcutaneously injected into C57BL / 6-WT mice. When tumor growth reached 150 mm ³ , mice were continuously fed a control solid diet or a solid feed containing GW3965 (150 mg / kg) and tumor growth was measured daily. n = 6 to 13. (GI) 5 × 10 ⁴ to mice given when normal solid feed or solid feed supplemented with GW3965 (100 mg / kg) forms tumors with a volume of 5 mm ^{3-10 mm 3 .} Total survival of mice after subcutaneous transplantation of B16F10 cells (G), 1 × 10 ⁶ MeWo cells (H) and 7.5 × 10 ⁵ SK-Mel-334.2 cells (I). n = 6-9 (F), 4-7 (H), 3-6 (I). Mouse endothelial cell antigen MECA in subcutaneous melanoma tumors formed by 1 × 10 ⁶ MeWo human melanoma cells in response to mouse treatment for 35 days with (JL) control diet or GW3965 supplemented diet (20 mg / kg) Tumor endothelial cell density (J) determined by immunohistochemical staining for -32, tumor cell proliferation (K) determined by staining for the proliferative marker Ki-67, and staining for truncated caspase-3. Tumor cell apoptosis (L) determined by. n = 5. Tumor volume was calculated as (minimum diameter) ² x (maximum diameter) / 2. All data are expressed as mean ± SEM. Scale bar, 5 mm (A to D), 50 μm (J, K), 25 μm (L). LXRβ agonism suppresses melanoma tumor growth (which is associated with FIGS. 21 (A)). (A) Weight measurement of mice fed with a control diet or a diet supplemented with GW3965 (20 mg / kg / day or 100 mg / kg / day) or a diet supplemented with T0901317 (20 mg / kg) for 65 days. n = 5-6. LXR agonism suppresses melanoma metastasis to the lungs and brain. (A) MeWo cells were pretreated with DMSO or GW3965 (1 μM) for 48 hours, and 4 × 10 ⁴ cells were intravenously injected into NOD Scid mice via the tail vein. Colonization in the lungs was monitored by weekly bioluminescence imaging. Representative H & E stained lungs corresponding to the last day (d70) are shown. n = 4-5. (BC) Control solid feed or solid feed containing GW3965 (20 mg / kg) or T0901317 (20 mg / kg) was intravenously infused into NOD Scid mice given starting 10 days prior to cancer cell infusion. Bioluminance imaging of lung metastases with ⁴ × 10 MeWo cells. A typical H & E stained lung corresponds to the final day of imaging. n = 5-6. (BC) Control solid feed or solid feed containing GW3965 (20 mg / kg) or T0901317 (20 mg / kg) was intravenously infused into NOD Scid mice given starting 10 days prior to cancer cell infusion. Bioluminance imaging of lung metastases with ⁴ × 10 MeWo cells. A typical H & E stained lung corresponds to the final day of imaging. n = 5-6. (F) After intracardiac injection of 1 × 10 ⁵ MeWo brain metastatic derivative cells into thymic nude mice given starting 0 days after infusion with a control diet or GW3965 supplement diet (100 mg / kg). Whole body and brain photon bundles in. n = 7. (G) Schematic of an experimental orthotopic metastasis model used to assess whether GW3965 treatment can suppress lung metastases after tumor resection. (H) After excision of a size-matched (about 300 mm ³ in volume) subcutaneous melanoma tumor formed by 1 × 10 ⁶ MeWo melanoma cells by a control diet or a diet containing GW3965 (100 mg / kg). Exvivo lung photon bundles as determined by bioluminescence imaging in NOD Scid mice fed over 1 month. Representative lungs stained for human vimentin are also shown. n = 7-9. (I) 4 × 10 ⁴ MeWo cells were intravenously injected into NOD Scid mice. After the onset of metastasis (which was detected by bioluminescence imaging at d42), mice were fed a control diet or GW3965 diet (100 mg / kg) as indicated for colony formation in the lungs. Progress was measured weekly. n = 6. (J) H & E-stained lungs removed on final day (d77) from NOD Scid mice fed a control diet or a diet supplemented with GW3965 (100 mg / kg) as shown in (I). Number of macroscopic metastatic nodules in. n = 4-5. (K) Intravenous injection of 4 × 10 ⁴ MeWo cells into NOD-Scid mice given a control solid feed or a GW3965 supplemented solid feed (20 mg / kg) starting 10 days prior to cancer cell infusion and continuously fed. Survival of all mice after. n = 5-6. All data are expressed as mean ± SEM. Suppression of genetically driven melanoma progression by LXR activation therapy. (A) Overall survival of Tyr :: CreER; Braf ^{V600E / +} ; Pten ^{lox / +} C57BL / 6 mice after systemic melanoma induction by intraperitoneal administration of 4-HT (25 mg / kg) for 3 consecutive days. After the first 4-HT injection, mice were randomly assigned to a control diet or a diet supplemented with GW3965 (100 mg / kg). n = 10-11. (B) Control solid feed or solid feed supplemented with GW3965 (100 mg / kg) given at the time of melanoma induction as described in (A) Tyr :: CreER; Braf ^{V600E / +} ; Pten ^lox Melanoma tumor loading expressed as a percentage of dorsal skin area measured on day 35 in ^{/ lox} mice. n = 4-5. (C) Control solid feed, or solid feed containing GW3965 (100 mg / kg), given after the overall induction of melanoma progression as described in (A) Tyr :: CreER; Braf ^{V600E / +} ; Number of grossly metastatic nodules to salivary gland lymph nodes detected after death in Pten ^{lox / lox} mice. n = 7-8. (D) Tumor growth after subcutaneous injection of 1 × 10 ⁵ Braf ^{V600E / +} ; Pten ^{− / −} ; CDKN2A ^{− / −} primary melanoma cells into syngeneic C57BL / 6-WT mice. When tumor growth reached 5 mm ^{3-10 mm 3 in} volume, mice were fed a control solid diet or a solid feed supplemented with GW3965 (100 mg / kg). n = 16-18. (E) 1 × 10 ⁵ Braf ^{V600E / +} ; Pten ^{− / −} ; CDKN2A ^{− / −} melanoma cells are subcutaneously injected, and the tumor growth reaches 5 mm ³ to 10 mm ³ in volume, and then the GW3965 diet (GW3965 diet). Overall survival of C57BL / 6-WT mice treated with 100 mg / kg) or a control diet. n = 7-8. (F) Colonization in the lung by 1 × 10 ⁵ Braf ^{V600E / +} ; Pten ^{− / −} ; CDKN2A ^{− / −} primary melanoma cells injected intravenously into C57BL / 6-WT mice. Immediately after cancer cell infusion, mice were randomly assigned to a control diet or GW3965 supplement diet (100 mg / kg) for the rest of the experiment. n = 14 to 15. All data are expressed as mean ± SEM. Scale bar, 2 mm (B), 5 mm (D). LXR-mediated inhibition of melanoma progression in a genetically driven melanoma mouse model (which is associated with FIGS. 24 (AC)). (A) Overall survival of Tyr :: CreER; Braf ^{V600E / +} ; Pten ^{lox / lox} C57BL / 6 mice after systemic melanoma induction by intraperitoneal administration of 4-HT (25 mg / kg) for 3 consecutive days. After the first 4-HT injection, mice were randomly assigned to a control diet or a diet supplemented with GW3965 (100 mg / kg). n = 7. (B) Tyr :: CreER; Braf ^{V600E / +} ; Pten ^{lox / lox} C57BL / to which a control diet or a GW3965 supplement diet (100 mg / kg) is taken 43 days after the induction of melanoma by intraperitoneal 4-HT administration. Typical image of 6 mice. List of 50 most upregulated genes in MeWo human melanoma cells in response to GW3965 treatment. Activation of LXRβ induces ApoE expression in melanoma cells; ApoE mediates LXRβ-dependent inhibition of the melanoma progression phenotype in vitro. (A-C) MeWo human melanoma cells (A), HT-144 human melanoma cells (B) and WM-266-4 human melanoma cells (C) were treated with GW3965 or T0901317 at the indicated concentrations for 48 hours. , ApoE expression levels were analyzed by qRT-PCR. n = 3. (D) Extracellular ApoE protein levels quantified by ELISA in serum-free conditioned medium collected from HT-144 human melanoma cells treated with DMSO, GW3965 or T0901317 at 1 μM for 72 hours. n = 3-4. (EF) ApoE neutralizing antibody (EF) ⁴ HT-144 cells treated with DMSO, GW3965 or T0901317 at 1 μM for 72 hours to each transwell at 40 μg / mL at the start of the assay. Cell infiltration phenotypes (E) and endothelial mobilization phenotypes (F) were tested in the presence of 1D7) or IgG control antibodies. n = 4. (GH) 1 × 10 ⁵ and 5 × 10 expressing control shRNA, or shRNA targeting ApoE, and treated with DMSO or GW3965 at 1 μM for 72 hours prior to each assay. Cell infiltration (G) and endothelial recruitment (F), respectively, brought about by the ^four MeWo cells. n = 7-8. (IJ) MeWo cells (I) and HT-144 cells transduced with control shRNA or NXRα or LXRβ targeting shRNA and subsequently treated with DMSO, GW3965 or T0901317 at 1 μM for 48 hours. Relative ApoE expression quantified by qRT-PCR in (J). n = 3-9. (K) In serum-free acclimatization medium collected from HT-144 cells transduced with control shRNA, or NXRα or LXRβ-targeting shRNA, and subsequently treated with DMSO or GW3965 at 1 μM for 72 hours. Extracellular ApoE protein levels as measured by ELISA. n = 3. All data are expressed as mean ± SEM. Scale bar, 50 μm. Activation of LXRβ suppresses melanoma infiltration and endothelial recruitment by transcriptionally enhancing ApoE expression in melanoma cells. (A) Driven from the ApoE promoter fused downstream of the multi-enhancer element 1 (ME.1) sequence or multi-enhancer element 2 (ME.2) sequence and treated with DMSO, GW3965 or T0901317 at 1 μM for 24 hours. Luciferase activity transfected into MeWo cells. n = 4-8. (B) Extracellular ApoE protein levels were quantified by ELISA in serum-free conditioned medium collected from MeWo cells treated with DMSO, GW3965 or T0901317 at 1 μM for 72 hours. n = 3-4. (C) Cell infiltration by 1 × 10 ⁵ MeWo cells pretreated with DMSO, GW3965 or T0901317 at 1 μM for 72 hours. At the start of the assay, ApoE neutralizing antibody (1D7) or IgG control antibody was added to each transwell at 40 μg / mL as shown. n = 7-8. (D) Presence of 1D7 or IgG antibody at 40 μg / mL of 5 × 10 ⁴ MeWo cells and 1 × 10 ⁵ endothelial cells pretreated with DMSO, GW3965 or T0901317 at 1 μM for 72 hours. Tested for their ability to mobilize below. n = 6-8. (E) Extracellular ApoE protein levels quantified by ELISA in serum-free conditioned medium from SK-Mel-334.2 primary human melanoma cells treated with DMSO or GW3965 at 1 μM for 72 hours. n = 4. (FG) 5 × 10 ⁴ SK-Mel-334.2 cells pretreated with GW3965 at 1 μM for 72 hours in a cell infiltration assay in the presence of 1D7 or IgG antibody at 40 μg / mL. It was subjected to (F) and the endothelial mobilization assay (G). n = 7-8. (H) ME. 1 Enhancer element or ME. The activity of the ApoE promoter fused to the two enhancer elements was measured in the presence of DMSO or GW3965 (1 μM) for 24 hours in control shRNA, or luciferase reporter activity in MeWo cells expressing shRNA targeting LXRα or LXRβ. Asked by doing. n = 3-8. (I) Human MeWo expressing control shRNA, or LXRα or LXRβ targeting shRNA, in response to 72 hours of treatment with extracellular ApoE protein levels quantified by ELISA in response to treatment with GW3965 or T0901317 (1 μM). Evaluated in serum-free conditioned medium collected from melanoma cells. n = 3-8. All data are expressed as mean ± SEM. Scale bar, 50 μm. Therapeutic delivery of LXR agonists upregulates melanoma-derived ApoE expression and systemic ApoE expression. (AB) ApoE expression levels quantified by qRT-PCR in subcutaneous tumors formed by B16F10 mouse melanoma cells injected into C57BL / 6 mice. After the formation of a 5 mm ³ tumor, the mice were fed a control diet, a diet containing GW3965 (20 mg / kg) (A), or a diet containing T0901317 (20 mg / kg) (B). Gave for days. n = 3-4. Primary tumors (C), lungs formed by MeWo human melanoma cells transplanted into NOD Scid mice fed (CE) control solid feed or solid feed supplemented with GW3965 (20 mg / kg). ApoE transcript expression in metastases (D) and brain metastases (E). ApoE levels were assessed on days 35 (C), 153 (D) and 34 (E) after cancer cell injection. n = 3-5. (F) Relative expression levels of LXRα, LXRβ and ApoE are targeted to control hairpins or ThenRNA (sh_mLXRα) targeting mouse LXRα, shRNA (sh_mLXRβ) targeting mouse LXRβ, or mouse ApoE. It was determined by qRT-PCR in B16F10 mouse melanoma cells expressing shRNA (sh_mApoE). (GH) ApoE mRNA levels (G) and ABCA1 mRNA levels (H) as measured by qRT-PCR in B16F10 cells expressing control shRNA or shRNA targeting mouse LXRβ or mouse ApoE. Cells were treated with DMSO or GW3965 at 5 μM for 48 hours. n = 3. (I) The mRNA level of ABCA1 measured by qRT-PCR in systemic leukocytes taken from LXRα − / − mice or LXRβ − / − mice fed a control diet or GW3965 supplement diet (20 mg / kg) for 10 days. n = 3-4. (J) Relative expression of ApoE mRNA, expressed as the frequency of SAGE tags, in mouse skin and lung tissues is publicly available from the Cancer Genome Database Project (CGAP) funded by NCI. It was obtained using the existing mSAGE Expression Matrix database. (K) Relative expression of ApoE mRNA as determined by qRT-PCR in MeWo melanoma cells (LM2) isolated from lung metastatic nodules, or in primary tumors, relative to non-selected MeWo parent cells of the control. n = 3. LXRβ agonism suppresses melanoma tumor growth and metastasis by inducing melanoma-derived ApoE expression and systemic ApoE expression. (A) Adipose tissue, lung tissue and brain tissue lysates taken from wild-type mice fed a control solid diet or a solid feed supplemented with GW3965 (20 mg / kg) or T0901317 (20 mg / kg) for 10 days. Western blot measurements of ApoE protein levels in. (B) Quantification of ApoE protein expression based on the Western blot shown in (A). Total tubulin was used as an endogenous control for normalization. n = 3-5. (C) The expression level of ApoE determined by qRT-PCR in systemic leukocytes obtained from mice fed a control diet or a diet supplemented with GW3965 or T0901317 at 20 mg / kg for 10 days. n = 3-6. (D) B16F10 control cells, or B16F10 cells expressing shRNA (sh_mLXRα) targeting mouse LXRα, or shRNA (sh_mLXRβ) targeting mouse LXRβ, were used in C57BL / 6-WT mice, LXRα-/-. Subcutaneous injection into mice or LXRβ − / − mice. When the tumor reached 5 mm ^{3-10 mm 3 in} volume, the mice were fed a control diet or a diet supplemented with GW3965 (20 mg / kg) for 7 days, after which the final tumor volume was measured. Representative tumor images taken at the end point are shown in the right panel. n = 6-18. (E) ApoE transcript levels quantified by qRT-PCR in systemic leukocytes removed from LXRα − / − mice or LXRβ − / − mice fed a control diet or GW3965 supplement diet (20 mg / kg) for 10 days. n = 3-5. (F) Subcutaneous tumor growth by B16F10 cells expressing 5 × 10 ⁴ B16F10 control cells or shRNA (sh_mApoE) targeting mouse ApoE in C57BL / 6-WT mice or ApoE − / − mice. After tumor formation with a volume of 5 mm ^{3-10 mm 3 ,} mice were fed a control diet or a diet supplemented with GW3965 (20 mg / kg) for 7 days to quantify the final tumor volume. .. A representative image of the tumor taken out on the last day of measurement (d12) is shown on the right. n = 8-18. (G) Colonization in the lung by 5 × 10 ⁴ B16F10 cells transduced by control shRNA or shRNA or sh_mApoE and intravenously injected into C57BL / 6-WT or ApoE − / − mice. Starting 10 days prior to cancer cell infusion, mice were assigned to treatment with a control diet or treatment with a GW3965 supplement diet (20 mg / kg). Lung metastases were quantified at d22 by bioluminescence imaging. Representative lungs removed at the end point (d22) are shown in the right panel. n = 5-10. (H) ApoE protein expression determined by blind immunohistochemical analysis in non-metastatic (n = 39) and metastatic (n = 34) primary melanoma skin lesion samples obtained from patients in MSKCC. .. The proportion of ApoE-positive stained cell area was quantified as a percentage of total tumor area. (I) Kaplan-Meier curve for the MSKCC cohort (n = 71) showing metastasis-free survival of a patient as a function of ApoE protein expression in the patient's primary melanoma lesion. Melanomas with ApoE levels above the population median were classified as ApoE positive (pos), whereas tumors with ApoE expression below this median were classified as ApoE negative (neg). All data are expressed as mean ± SEM. Scale bar, 5 mm (D and F), 100 μm (H). Activation of LXRβ suppresses in vivo growth of melanoma strains that are resistant to dacarbazine and vemurafenib. (A) In vitro cell growth by 2.5 × 10 ⁴ B16F10 parent cells and in vitro-derived B16F10 DTIC-resistant cells in response to various doses of dacarbazine (DTIC) added to the cell medium for 4 days. n = 3. (B-D) Tumor growth by 5 × 10 ⁴ DTIC-sensitive B16F10 parent cells (B) or 5 × 10 ⁴ DTIC-resistant B16F10 cells (C) injected subcutaneously into C57BL / 6-WT mice. After tumor growth reaches 5 mm ^3-10 mm ³ by volume, mice are treated with dacarbazine (50 mg / kg, ip, daily) or control vehicle and with regular solid feed or GW3965 (100 mg / kg). Was randomly assigned to the solid feed supplemented with. The tumor volume measurement result on the final day is shown in (D). n = 8 to 16 (B), 7 to 8 (C). (EF) Tumor growth by DTIC-sensitive MeWo parent cells and in vivo-derived DTIC-resistant MeWo human melanoma cells in response to DTIC or GW3965 treatment. 5 × 10 ⁵ cells were subcutaneously injected into NOD Scid gamma mice. After tumor formation with a volume of 5 mm ^{3-10 mm 3 ,} mice are blindly controlled or treated with DTIC (50 mg / kg, ip, with a 2-day washout interval 5). Administered daily in a daily cycle) or assigned to GW3965 supplemental diet treatment (100 mg / kg). The tumor measurement results on the final day are shown in (F). n = 6-8. (G) 2 × subcutaneously injected into NOD Scid gamma mice assigned to a control diet or a diet supplemented with GW3965 (100 mg / kg) after the tumor had grown to 5 mm ^{3-10 mm 3 in} volume. Tumor growth with 10 ⁶ SK-Mel-239 vemurafenib resistant cloned cells. n = 7-8. (H) Survival of all mice after transplantation of 2 × 10 ⁶ SK-Mel-239 vemurafenib resistant cells. As the tumor grew to 5 mm ^{3-10 mm 3 in} volume, the mice were continuously fed a control diet or a diet supplemented with GW3965 (100 mg / kg). n = 7. (I) An experimentally derived model showing the involvement of systemic ApoE and melanoma autonomous ApoE by LXRβ activation therapy in mediating suppression of the melanoma progression phenotype. Extracellular ApoE coordinately inhibits melanoma cell infiltration and cell non-autonomous endothelial recruitment through targeting the LRP1 receptor on melanoma cells and the LRP8 receptor on endothelial cells, respectively. To suppress. All data are expressed as mean ± SEM. Scale bar, 5 mm. Inhibition of dacarbazine induction of tumor growth by human melanoma cells. (A) Tumor growth by 5 × 10 ⁵ DTIC-sensitive MeWo parent cells subcutaneously injected into Nod SCID gamma mice. When the tumor reaches a volume of 5 mm ^{3-10 mm 3 ,} mice are treated with a control vehicle or DTIC (50 mg / kg, ip., Administered daily in a 5-day cycle with a 2-day washout interval). The tumor volume was measured twice a week. n = 6. ApoE-mediated suppression of cell infiltration across many cancer types. (AB) 5 × 10 ⁴ MUM2B human melanoma cells and OCM1 human melanoma cells, (CE) 5 × 10 ⁴ MDA-231 human triple negative breast cancer cells, MDA-468 human triple Negative breast cancer cells and BT549 human triple negative breast cancer cells, (FG) 5 × 10 ⁴ PANC1 human pancreatic cancer cells and BXPC-3 human pancreatic cancer cells, and (HI) 5 × 10 ⁴ 786 -00 human kidney cancer cells and RCC4 human kidney cancer cells were tested in vitro for their ability to infiltrate through Matrigel-coated transwell inserts. BSA and recombinant ApoE were added to the cell medium at 100 μg / mL at the start of the assay. n = 4. All data are expressed as mean ± SEM; ^* p <0.05, ^** p <0.01, ^*** p <0.001. Effect of LXR agonists (LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) and SB7422881) on ApoE expression in human melanoma cells. (A-D) MeWo human melanoma cells in 500 nM by DMSO or LXR agonists LXR-623 (A), WO-2007-002563 (B), WO-2010-0138598 (C) or SB7422881 (D). Treatment was performed at 1 μM or 2 μM for 48 hours. The expression level of ApoE was then quantified by qRT-PCR. n = 3. All data are expressed as mean ± SEM. ^* P <0.05, ^** p <0.01. Treatment with the LXR agonist GW3965 inhibits in vitro tumor cell infiltration of kidney, pancreatic and lung cancers. (A-C) 5 × 10 ⁴ RCC human kidney cancer cells (A), 5 × 10 ⁴ PANC1 human pancreatic cancer cells (B) treated with DMSO at 1 μM or GW3965 for 72 hours prior to assay. And Matrigel infiltration in transwell by 5 × 10 ⁴ H460 human lung cancer cells (C). n = 4. All data are expressed as mean ± SEM. ^* P <0.05, ^** p <0.01. Treatment with the LXR agonist GW3965 inhibits breast cancer tumor growth in vivo. Primary tumor growth with 2 × 10 ⁶ MDA-468 human breast cancer cells injected into the breast fat pad of NOD Scid gamma mice. Two days prior to cancer cell infusion, mice were assigned to a control diet treatment or diet supplemented with GW3965 (75 mg / kg) and maintained on the corresponding diet throughout the experiment. n = 8. All data are expressed as mean ± SEM. ^*** p <0.001. Effect of LXR agonists (LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) and SB7422881) on the melanoma progression phenotype in vitro. (A) 1 × 10 ⁵ MeWo pretreated with DMSO, LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) or SB7422881 at 1 μM for 72 hours each. Cell infiltration by human melanoma cells. The number of cells infiltrating the base side of the Matrigel-coated transwell insert was quantified. n = 5. (B) 5 × 10 ⁴ MeWo pretreated with DMSO, LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) or SB7422881 at 1 μM for 72 hours each. Endothelial mobilization by cells. Cancer cells were seeded on the bottom of a 24-well plate. Endothelial cells were seeded into transwell inserts attached to each well and migrated towards cancer cells. The number of endothelial cells migrating to the base side of each transwell insert was quantified. n = 4-5. All data are expressed as mean ± SEM. ^* P <0.05, ^** p <0.01. Effect of LXR agonists (LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) and SB7422881) on tumor growth in vivo. (A to D) Tumor growth by 5 × 10 ⁴ B16F10 mouse melanoma cells injected subcutaneously into 7-week-old C57BL / 6 mice. After the tumor reached 5 mm ^3-10 mm ³ in volume, mice were randomly treated with a control diet, LXR-623 supplemental diet at 20 mg / kg / day (A), WO- at 100 mg / kg / day. 2007-002563 (Ex.19) Supplemental food treatment (B), WO-2010-0138598 (Ex.19) supplementary food treatment (C) at 10 mg / kg / day or 100 mg / kg / day, or 100 mg / kg Assigned to SB742881 supplemental dietary treatment (D) at / day. n = 8-10. All data are expressed as mean ± SEM.

Detailed description of the invention

The present invention features methods for preventing or reducing the abnormal proliferation, differentiation or survival of cells. For example, the compounds of the invention do not increase the tumor in size or reach a metastatic state, or in reducing the risk of reaching a metastatic state, or the tumor does not grow in size or reach a metastatic state. May be useful in that. The subject compounds of the invention may be administered to stop the progression or development of cancer. In addition, the invention includes using the subject compounds of the invention to reduce the risk of cancer recurrence or to prevent cancer recurrence.

Metastatic progression requires the consistent expression of a variety of effector proteins involved in a common cell phenotype (Gupta and Massage, 2006, Cell, 127, 679-695; Hanahan and Weinberg). , 2011, Cell, 144, 646-674; Talmadge and Fiddler, 2010, Cancer Res., 70, 5649-5669; Hynes, 2003, Cell, 113, 821-823). Such coordinated expression states are evident in the gene expression profile of metastatic primary breast cancer (Wang et al., 2005, Lancet, 365, 671-679), as well as various exhibiting enhanced metastatic activity. It is evident in the profile of human cancer cell clones (Kang et al., 2003, Cancer Cell, 3, 537-549; Minn et al., 2005, Nature, 436, 518-524). In recent years, post-transcriptional regulation has emerged as a broad and robust mode of coordinated expression and phenotypic level regulation. The most studied class of post-transcriptional regulators with metastatic activity is small non-coding RNAs (miRNAs) (Bartel, 2009, Cell, 136, 215-233; Fabian et al., 2010, Annu. Rev. Biochem. , 79, 351 to 379; Filipowerz et al., 2008, Nat. Rev. Genet., 9, 102 to 114). Metastasis-promoting factor miRNA (Ma et al., 2007, Nature, 449, 682-688; Huang et al., 2008, Nat. Cell Biol., 10, 202-210) and metastasis-suppressing factor miRNA (Tavazoie et al., 2008, Nature, 451. 147-152) was first discovered in breast cancer. Subsequent studies have revealed more miRNAs that have a regulatory role in tumorigenesis and metastasis of other cancer types (Hatziapostolou et al., 2011, Cell, 147, 1233-1247; Hurst et al., 2009, Cancer Res. , 69, 7495-7298; Olson et al., 2009, Genes Dev., 23, 2152-2165; Zhang et al., 2010, Oncogene, 29, 937-948). In many cases, the expression levels of these miRNAs in human cancer samples support their experimental role in metastasis. Thus, deregulated miRNA expression (Garzon et al., 2010, Nat. Rev. Drag Discov., 9, 775-789; Lujambio and Lowe, 2012, Nature, 482, 347-355), and more recently, Deregulated expression of long non-coding RNAs (Calin et al., 2007, Nat. Rev. Cancer, 6, 857-866; Gupta et al., 2010, Nature, 464, 1071-176; Guttman et al., 2009, Nature, 458, 223 to 227; Huarte et al., 2010, Cell, 142, 409 to 419; Rower et al., 2010, Nat. Genet., 42, 1113 to 1117), as well as competing non-codes for binding of endogenous miRNAs. Deregulated expression of pseudogenes (Poliseno et al., 2010, Nature, 465, 1033-1038) appears to be a widespread feature of human cancer. Various clues about the robust regulation of specific miRNAs on metastatic progression were that the coordinated targeting of multiple metastatic genes by only one metastasis-suppressing factor, miRNA, was involved in the dramatic metastasis-suppressing effect. (Tavazoie et al., 2008, Nature, 451 and 147-152). It is becoming clear that such a wide variety of gene targeting with various miRNAs is a characteristic feature of these regulators.

At the conceptual level, the need for a wide range of regulation of gene expression in cancer is easily understood. MiRNAs may be able to exert robust metastasis inhibition due to their ability to target the large number of genes required for metastasis. It appears that silencing of miRNAs via genetic or metastatic mechanisms will facilitate cancer progression by desuppressing a number of metastatic promoters (Png et al., 2011, Nature,
481, 190-194). The role for the convergent regulation of only one gene by a large number of metastasis-regulating miRNAs is further dominated. This scenario would emerge if there were important genes that acted as robust inhibitors of metastatic progression. Convergent and collaborative targeting of this gene with multiple miRNAs may achieve maximum silencing of such important metastasis-suppressing factor genes. This scenario may be observed when complete loss of the target gene may not be tolerated by the cell, as opposed to genetic deletion, where the gene is, for example, to mediate metabolic effects. It seems that it will be required at a low level. Given this possibility, the search for collaborative metastasis-promoting factor miRNAs may discover novel genes that are very important for metastasis suppression and are more effective for metastasis prevention. It may provide therapeutic insights into the procedure.

As disclosed herein, through a systematic in vivo selection-based approach, a set of miRNAs is a melanoma, i.e., a highly widespread cancer with increasing incidence (Garbe and). It was confirmed that it is deregulated in a large number of unrelated metastatic strains derived from a large number of patients with Leiter, 2009, Clin. Dermatol., 27, 3-9). As disclosed herein, miR-1908, miR-199a-3p and miR-199a-5p are robust to melanoma transfer through the convergent targeting of the metabolic genes ApoE and the heat shock protein DNAJA4. It acts as an endogenous promoter. Loss-of-function analysis, gain-of-function analysis, and epistasis analysis outline a collaborative miRNA network that maximally silences ApoE signaling. ApoE secreted by cancer cells inhibits metastatic infiltration and endothelial recruitment, which is mediated by its action on different receptors in melanoma cells and endothelial cells. These miRNAs show a significant prognostic potential in identifying patients who develop metastatic recurrence of melanoma, while therapeutic delivery of LNAs targeting these miRNAs significantly inhibits melanoma metastasis. .. The current lack of effective treatments to prevent melanoma metastasis after surgical resection (Garbe et al., 2011, Oncologist, 16, 5-24) is a molecule with improved metastatic progression of melanoma. It requires a proper and mechanical understanding. To this end, the findings disclosed herein reveal several important novel non-coding and coding genes involved in melanoma progression and are also at risk for melanoma metastasis. Brings new means for both identifying large patients and treating such patients.

Below are the nucleic acid and amino acid sequences of the members of the network mentioned above as well as some other sequences.

ＡＰＯＥ－アミノ酸配列（配列番号２）

（下線が施された残基１３６～残基１５０はＡｐｏＥのＬＲＰ結合ドメインを表す）

ＤＮＡＪＡ４イソ型１－ＲＮＡ配列（配列番号３）

ＤＮＡＪＡ４イソ型１－アミノ酸配列（配列番号４）

ＤＮＡＪＡ４イソ型２－ＲＮＡ配列（配列番号５）

ＤＮＡＪＡ４イソ型２－アミノ酸配列（配列番号６）

ＤＮＡＪＡ４イソ型３－ＲＮＡ配列（配列番号７）

ＤＮＡＪＡ４イソ型３－アミノ酸配列（配列番号８）

ＬＲＰ１－ＲＮＡ配列（配列番号９）

ＬＲＰ１－アミノ酸配列（配列番号１０）

ＬＲＰ８イソ型１－ＲＮＡ配列（配列番号１１）

ＬＲＰ８イソ型１－アミノ酸配列（配列番号１２）

ＬＲＰ８イソ型２－ＲＮＡ配列（配列番号１３）

ＬＲＰ８イソ型２－アミノ酸配列（配列番号１４）

ＬＲＰ８イソ型３－ＲＮＡ配列（配列番号１５）

ＬＲＰ８イソ型３－アミノ酸配列（配列番号１６）

ＬＲＰ８イソ型４－ＲＮＡ配列（配列番号１７）

ＬＲＰ８イソ型４－アミノ酸配列（配列番号１８）

ＣＴＧＦ－ＲＮＡ配列（配列番号１９）

ＣＴＧＦ－アミノ酸配列（配列番号２０）

ＬＸＲ－ａイソ型１：ＲＮＡ配列（配列番号２１）

ＬＸＲ－ａ（ＮＲ１Ｈ３）イソ型１：アミノ酸配列（配列番号２２）

ＬＸＲ－ａ（ＮＲ１Ｈ３）イソ型２：ＲＮＡ配列（配列番号２３）

ＬＸＲ－ａ（ＮＲ１Ｈ３）イソ型２：アミノ酸配列（配列番号２４）

ＬＸＲ－ａ（ＮＲ１Ｈ３）イソ型３：ＲＮＡ配列（配列番号２５）

ＬＸＲ－ａ（ＮＲ１Ｈ３）イソ型３：アミノ酸配列（配列番号２６）

ＬＸＲ－ａ（ＮＲ１Ｈ３）イソ型４：ＲＮＡ配列（配列番号２７）

ＬＸＲ－ａ（ＮＲ１Ｈ３）イソ型４：アミノ酸配列（配列番号２８）

ＬＸＲ－ｂ（ＮＲ１Ｈ２）イソ型１：ＲＮＡ配列（配列番号２９）

ＬＸＲ－ｂ（ＮＲ１Ｈ２）イソ型１：アミノ酸配列（配列番号３０）

ＬＸＲ－ｂ（ＮＲ１Ｈ２）イソ型２：ＲＮＡ配列（配列番号３１）

ＬＸＲ－ｂ（ＮＲ１Ｈ２）イソ型２：アミノ酸配列（配列番号３２）

ｈａｓ－ｍｉＲ－１９９ａ－１配列（配列番号３３）
GCCAACCCAGUGUUCAGACUACCUGUUCAGGAGGCUCUCAAUGUGUACAGUAGUCUGCACAUUGGUUAGGC

ｈａｓ－ｍｉＲ－１９９ａ－２配列（配列番号３４）
AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCCAGUGUUCAGACUACCUGUUCAGGACAAUGCCGUUGUACAGUAGUCUGCACAUUGGUUAGACUGGGCAAGGGAGAGCA

ｈａｓ－ｍｉＲ－１９０８配列（配列番号３５）
CGGGAAUGCCGCGGCGGGGACGGCGAUUGGUCCGUAUGUGUGGUGCCACCGGCCGCCGGCUCCGCCCCGGCCCCCGCCCC

ｈａｓ－ｍｉＲ－７－１配列（配列番号３６）
UUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAGUGAUUUUGUUGUUUUUAGAUAACUAAAUCGACAACAAAUCACAGUCUGCCAUAUGGCACAGGCCAUGCCUCUACAG

ｈａｓ－ｍｉＲ－７－２配列（配列番号３７）
CUGGAUACAGAGUGGACCGGCUGGCCCCAUCUGGAAGACUAGUGAUUUUGUUGUUGUCUUACUGCGCUCAACAACAAAUCCCAGUCUACCUAAUGGUGCCAGCCAUCGCA

ｈａｓ－ｍｉＲ－７－３配列（配列番号３８）
AGAUUAGAGUGGCUGUGGUCUAGUGCUGUGUGGAAGACUAGUGAUUUUGUUGUUCUGAUGUACUACGACAACAAGUCACAGCCGGCCUCAUAGCGCAGACUCCCUUCGAC

ｍｉＲ－Ｚｉｐ １９９ａ－３ｐ配列（配列番号３９）
GATCCGACAGTAGCCTGCACATTAGTCACTTCCTGTCAGTAACCAATGTGCAGACTACTGTTTTTTGAATT

ｍｉＲ－Ｚｉｐ １９９ａ－５ｐ配列（配列番号４０）
GATCCGCCCAGTGCTCAGACTACCCGTGCCTTCCTGTCAGGAACAGGTAGTCTGAACACTGGGTTTTTGAATT

ｍｉＲ－Ｚｉｐ １９０８配列（配列番号４１）
GATCCGCGGCGGGAACGGCGATCGGCCCTTCCTGTCAGGACCAATCGCCGTCCCCGCCGTTTTTGAATT

ｍｉＲ－Ｚｉｐ ７配列（配列番号４２）
GATCCGTGGAAGATTAGTGAGTTTATTATCTTCCTGTCAGACAACAAAATCACTAGTCTTCCATTTTTGAATT

このネットワークのメンバーは、転移性メラノーマを処置するための標的として使用することができる。加えて、これらのメンバーは、対象が転移性メラノーマを有するかどうか、または、転移性メラノーマを有する危険性があるかどうかを決定するためのバイオマーカーとして、あるいは、そのような障害を有する患者の予後を明らかにするための、または、そのような障害を有する患者の監視のためのバイオマーカーとして使用することができる。したがって、本発明は、転移性メラノーマを、これらのメンバーの１つまたは複数を標的化することによって処置する方法、当該ガンを阻害するための治療療法の効力を明らかにする方法、および、抗ガン剤を特定する方法を包含する。また、対象が転移性メラノーマを有するかどうか、または、転移性メラノーマを有する危険性があるかどうかを診断する方法、および、当該障害を発症する危険性があると考えられる対象をスクリーニングする方法も提供される。本発明はまた、上述の方法を行うために好適である様々なキットを包含する。 APOE-RNA sequence (SEQ ID NO: 1)

APOE-amino acid sequence (SEQ ID NO: 2)

(Underlined residues 136-150 represent the LRP binding domain of ApoE)

DNAJA4 isotype 1-RNA sequence (SEQ ID NO: 3)

DNAJA4 isotype 1-amino acid sequence (SEQ ID NO: 4)

DNAJA4 isotype 2-RNA sequence (SEQ ID NO: 5)

DNAJA4 isotype 2-amino acid sequence (SEQ ID NO: 6)

DNAJA4 isotype 3-RNA sequence (SEQ ID NO: 7)

DNAJA4 isotype 3-amino acid sequence (SEQ ID NO: 8)

LRP1-RNA sequence (SEQ ID NO: 9)

LRP1-amino acid sequence (SEQ ID NO: 10)

LRP8 isotype 1-RNA sequence (SEQ ID NO: 11)

LRP8 isotype 1-amino acid sequence (SEQ ID NO: 12)

LRP8 isotype 2-RNA sequence (SEQ ID NO: 13)

LRP8 isotype 2-amino acid sequence (SEQ ID NO: 14)

LRP8 isotype 3-RNA sequence (SEQ ID NO: 15)

LRP8 isotype 3-amino acid sequence (SEQ ID NO: 16)

LRP8 isotype 4-RNA sequence (SEQ ID NO: 17)

LRP8 isotype 4-amino acid sequence (SEQ ID NO: 18)

CTGF-RNA sequence (SEQ ID NO: 19)

CTGF-amino acid sequence (SEQ ID NO: 20)

LXR-a isotype 1: RNA sequence (SEQ ID NO: 21)

LXR-a (NR1H3) Isotype 1: Amino acid sequence (SEQ ID NO: 22)

LXR-a (NR1H3) Isotype 2: RNA sequence (SEQ ID NO: 23)

LXR-a (NR1H3) isotype 2: amino acid sequence (SEQ ID NO: 24)

LXR-a (NR1H3) isotype 3: RNA sequence (SEQ ID NO: 25)

LXR-a (NR1H3) isotype 3: amino acid sequence (SEQ ID NO: 26)

LXR-a (NR1H3) isotype 4: RNA sequence (SEQ ID NO: 27)

LXR-a (NR1H3) isotype 4: amino acid sequence (SEQ ID NO: 28)

LXR-b (NR1H2) isotype 1: RNA sequence (SEQ ID NO: 29)

LXR-b (NR1H2) Isotype 1: Amino acid sequence (SEQ ID NO: 30)

LXR-b (NR1H2) Isotype 2: RNA sequence (SEQ ID NO: 31)

LXR-b (NR1H2) Isotype 2: Amino acid sequence (SEQ ID NO: 32)

has-miR-199a-1 sequence (SEQ ID NO: 33)
GCCAACCCAGUGUUCAGACUACCUGUUCAGGAGGCUCUCAAUGUGUACAGUAGUCUGCACAUUGGUUAGGC

has-miR-199a-2 sequence (SEQ ID NO: 34)
AGGAAGCUUCUGGAGAUCCUGCUCCGUCGCCCCAGUGUUCAGACUACCUGUUCAGGACAAUGCCGUUGUACAGUAGUCUGCACAUUGGUUAGACUGGGCAAGGGAGAGCA

has-miR-1908 sequence (SEQ ID NO: 35)
CGGGAAUGCCGCGGCGGGGACGGCGAUUGGUCCGUAUGUGUGGUGCCACCGGCCGCCGGCUCCGCCCCGGCCCCCGCCCC

has-miR-7-1 sequence (SEQ ID NO: 36)
UUGGAUGUUGGCCUAGUUCUGUGUGGAAGACUAGUGAUUUUGUUGUUUUAGAUAACUAAAUCGACAACAAAUCACAGUCUGCCAUAUGGCACAGGCCAUGCCUCUACAG

has-miR-7-2 sequence (SEQ ID NO: 37)
CUGGAUACAGAGUGGACCGGCUGGCCAUCUGGAAGACUAGUGAUUUUGUUGUUGUCUUACUGCGCUCAACAACAAAUCCCAGUCUACCUAAUGGUGCCAGCCAUCGCA

has-miR-7-3 sequence (SEQ ID NO: 38)
AGAUUAGAGUGGCUGUGGUCUAGUGCUGUGGAAGACUAGUGAUUUUGUUGUUCUGAUGUACUACGACAACAAGUCACAGCCGGCCUCAUAGCGCAGACUCCCUUCGAC

miR-Zip 199a-3p sequence (SEQ ID NO: 39)
GATCCGACAGTAGCCTGCACATTAGTCACTTCCTGTCAGTAACCAATGTGCAGACTACTGTTTTTTGAATT

miR-Zip 199a-5p sequence (SEQ ID NO: 40)
GATCCGCCCAGTGCTCAGACTACCCGTGCCTTCCTGTCAGGAACAGGTAGTCTGAACACTGGGTTTTTGAATT

miR-Zip 1908 sequence (SEQ ID NO: 41)
GATCCGCGGCGGGAACGGCGATCGGCCCTTCCTGTCAGGACCAATCGCCGTCCCCGCCGTTTGAATT

miR-Zip 7 sequence (SEQ ID NO: 42)
GATCCGTGGAAGATTAGTGAGTTTATTATCTTCCTGTCAGACAACAAAATCACTAGTCTTCCATTTTTGAATT

Members of this network can be used as targets for treating metastatic melanoma. In addition, these members serve as biomarkers to determine if a subject has or is at risk of having metastatic melanoma, or in patients with such disorders. It can be used to determine prognosis or as a biomarker for monitoring patients with such disorders. Accordingly, the present invention is a method of treating metastatic melanoma by targeting one or more of these members, a method of demonstrating the efficacy of therapeutic therapies to inhibit the cancer, and anti-cancer. Includes methods for identifying agents. There are also methods for diagnosing whether a subject has or is at risk of having metastatic melanoma, and for screening subjects who are considered to be at risk of developing the disorder. Provided. The invention also includes various kits suitable for performing the methods described above.

The ApoE polypeptide term "polypeptide or peptide", as used herein, is produced by recombination or synthesis of any of the above-mentioned metastasis-suppressing factors having a particular domain or moiety involved in the network. Includes fused or chimeric compounds. These terms also include analogs, fragments, extensions or derivatives of peptides (eg, those with added amino-terminal methionine that are useful for expression in prokaryotic cells).

"Apolypoprotein polypeptide or ApoE polypeptide", as used herein, is an analog, fragment, extension or extension of apolypoprotein that is a peptide between 10 and 200 amino acid residues in length. Means a peptide, drug or compound that mimics the function of a native apolypoprotein, whether in vivo or in vitro, including derivatives, such peptide being a natural or non-natural amino acid containing an amide bond. Both of these can be contained. Peptide fragments of apolipoproteins may be modified as known in the art to improve their stability or bioavailability in vivo, and may vary to amino acid side chains. It may contain an organic compound that is bound via a strong bond.

In one aspect, the present inventors have isolated apoEp1 having the amino acid sequence of TQQIRLQAEIFQAR (mouse) (SEQ ID NO: 43) or AQQIRLQAEAFQAR (human) (SEQ ID NO: 44). A method for using a B peptide, or an analog, fragment, extension or derivative of the peptide. The present invention also includes apoEp1. Includes nucleic acid molecules encoding B peptides or analogs, fragments, extensions or derivatives thereof.

The term "analog" refers to an amino acid that is substantially identical to a native peptide in which one or more residues of any peptide are conservatively replaced by functionally similar residues. Peptides with a residue sequence that include peptides that exhibit the ability to mimic native peptides. Examples of conservative substitutions include the use of one non-polar (hydrophilic) residue (eg, alanine, isoleucine, valine, leucine or methionine) in place of another non-polar (hydrophilic) residue. The use of one polar (hydrophilic) residue in place of another polar (hydrophilic) residue, eg, between arginine and leucine, between glutamine and aspartin, between glycine and serine, etc. Substitution, using one basic residue (eg, such as leucine, arginine or histidine) in place of another basic residue, or using one acidic group (eg, aspartic acid or glutamine) to another. Includes use in place of acidic residues.

The expression "conservative substitution" is also conditional on the use of chemically derivatized residues in place of non-derivatized residues, provided that such polypeptides exhibit essential activity. Included in. Analogs of the peptides include peptides having the following sequences: TAQIRLQAEIFQAR (SEQ ID NO: 45), TQAIRLQAEIFQAR (SEQ ID NO: 46), TQQARLQAEIFQAR (SEQ ID NO: 47) and TQQIALQAEIFQAR (SEQ ID NO: 48).

"Derivative" refers to a peptide in which one or more residues are chemically derivatized by a functional side chain reaction. In such derivatized molecules, for example, a free amino group forms an amine hydrochloride, a p-toluenesulfonyl group, a carbobenzoxyl group, a t-butyloxycarbonyl group, a chloroacetyl group or a formyl group. Includes such molecules that have been derivatized for. Free carboxyl groups may be derivatized to form salts, methyl esters and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included are such peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline may be used in place of proline; 5-hydroxylysine may be used in place of lysine; 3-methylhistidine may be used in place of histidine; Homoserine may be used in place of serine; orornitin may be used in place of lysine. The polypeptides of the invention also have one or more residues relative to the sequence of polypeptide shown herein, as long as the essential activity is maintained. Contains polypeptides with additions and / or deletions of.

The term "fragment" refers to a subject peptide having an amino acid residue sequence whose amino acid residue sequence is shorter than the amino acid residue sequence of the peptide shown herein, regardless of the subject peptide.

The term "extended" has an amino acid sequence that is longer than the amino acid sequence of the peptide of the invention (either at the carboxy terminus or at the amino terminus) by one or two amino acids in any subject peptide. The subject peptide is shown. Preferably, the extension is carried out at the amino terminus. Fragments and extensions of the peptides include peptides having the following sequences: QTQQIRLQAEIFQAR (SEQ ID NO: 49) and QQIRLQAEIFQAR (SEQ ID NO: 50).

Various ApoE polypeptides and methods of their preparation are described in US Pat. No. 6,652,860 (which is incorporated herein by reference).

LXR Agonists The methods of the invention can include administering LXR agonists for the prevention and treatment of metastasis. The LXR agonist can be a compound according to formula I, formula II, formula III or formula IV shown below.

Formula I is provided below:

またはその医薬的に許容される塩、但し、式Ｉにおいて、
Ａｒはアリール基である；
Ｒ^１は、－ＯＨ、－ＣＯ_２Ｈ、－Ｏ－（Ｃ_１～Ｃ_７）アルキル、－ＯＣ（Ｏ）－、－（Ｃ_１～Ｃ_７）アルキル、－Ｏ－（Ｃ_１～Ｃ_７）ヘテロアルキル、－ＯＣ（Ｏ）－（Ｃ_１～Ｃ_７）ヘテロアルキル、－ＮＨ_２、－ＮＨ（Ｃ_１～Ｃ_７）アルキル、－Ｎ（（Ｃ_１～Ｃ_７）アルキル）_２および－ＮＨ－Ｓ（Ｏ）_２（Ｃ_１～Ｃ_５）アルキルからなる群から選択されるものである；
Ｒ^２は、（Ｃ_１～Ｃ_７）アルキル、（Ｃ_１～Ｃ_７）ヘテロアルキル、アリールおよびアリール（Ｃ_１～Ｃ_７）アルキルからなる群から選択されるものである；
Ｘ^１、Ｘ^２、Ｘ^３、Ｘ^４、Ｘ^５およびＸ^６はそれぞれが独立して、Ｈ、（Ｃ^１～Ｃ^５）アルキル、（Ｃ^１～Ｃ^５）ヘテロアルキル、ＦおよびＣｌからなる群から選択されるものであり、但し、Ｘ^１～Ｘ^６のうちの最大でも３つが、Ｈ、（Ｃ^１～Ｃ^５）アルキル、（Ｃ^１～Ｃ^５）ヘテロアルキルである；かつ
Ｙは、－Ｎ（Ｒ^１２）Ｓ（Ｏ）_ｍ－、－Ｎ（Ｒ^１２）Ｓ（Ｏ）_ｍＮ（Ｒ^１３）－、－Ｎ（Ｒ^１２）Ｃ（Ｏ）－、－Ｎ（Ｒ^１２）Ｃ（Ｏ）Ｎ（Ｒ^１３）－、－Ｎ（Ｒ^１２）Ｃ（Ｓ）－および－Ｎ（Ｒ^１２）Ｃ（Ｏ）Ｏ－からなる群から選択される二価の連結基であり、
但し、Ｒ^１２およびＲ^１３はそれぞれが独立して、Ｈ、（Ｃ_１～Ｃ_７）アルキル、（Ｃ_１～Ｃ_７）ヘテロアルキル、アリールおよびアリール（Ｃ_１～Ｃ_７）アルキルからなる群から選択され、また、必要に応じて、Ｙが－Ｎ（Ｒ^１２）Ｓ（Ｏ）_ｍ－または－Ｎ（Ｒ^１２）Ｓ（Ｏ）_ｍＮ（Ｒ^１３）－であるときには、Ｒ^１２は、ＡｒまたはＲ^２に対する共有結合による結合を介してＡｒまたはＲ^２にそれぞれ融合する５員環または６員環を形成し、この場合、下付き添え字ｍは１～２の整数である；
但し、Ｒ^１がＯＨであり、かつ、－Ｙ－Ｒ^２が－Ｎ（Ｒ^１２）Ｓ（Ｏ）_ｍ－Ｒ^２または－Ｎ（Ｒ^１２）Ｃ（Ｏ）Ｎ（Ｒ^１３）－Ｒ^２であり、Ａｒに結合する第四級炭素に対してパラ位に結合するとき、および、Ｒ^２が、フェニル、ベンジルまたはベンゾイルであるときには、ｉ）Ｒ^１２またはＲ^１３の少なくとも１つが水素以外であり、かつ、電子吸引性置換基を含有し、あるいは、ｉｉ）Ｒ^２が、アミノ、アセトアミド、ジ（Ｃ_１～Ｃ_７）アルキルアミノ、（Ｃ_１～Ｃ_７）アルキルアミノ、ハロゲン、ヒドロキシ、ニトロまたは（Ｃ_１～Ｃ_７）アルキルとは異なる成分により置換され、あるいは、ｉｉｉ）Ｒ^２のベンゼン環部分が、Ｙ基に加えて、または、Ｙに対する結合点に加えて少なくとも３つの独立して選択された基により置換される。

Or its pharmaceutically acceptable salt, provided that in Formula I,
Ar is an aryl group;
R ¹ is -OH, -CO ₂ H, -O- (C ₁ to C ₇ ) alkyl, -OC (O)-,- (C ₁ to C ₇ ) alkyl, -O- (C ₁ to C ₇ ). ) Heteroalkyl, -OC (O)-(C ₁ to C ₇ ) heteroalkyl, -NH ₂ , -NH (C ₁ to C ₇ ) alkyl, -N ((C ₁ to C ₇ ) alkyl) ₂ and- It is selected from the group consisting of NH-S (O) ₂ (C ₁ to C ₅ ) alkyl;
R ² is selected from the group consisting of (C ₁ to C ₇ ) alkyl, (C ₁ to C ₇ ) heteroalkyl, aryl and aryl (C ₁ to C ₇ ) alkyl;
X ¹ , X ² , X ³ , X ⁴ , X ⁵ and X ⁶ each independently consist of H, (C ¹ to C ⁵ ) alkyl, (C ¹ to C ⁵ ) heteroalkyl, F and Cl. It is selected from the group, provided that at most three of X ¹ to X ⁶ are H, (C ¹ to C ⁵ ) alkyl, (C ¹ to C ⁵ ) heteroalkyl; and Y is. , -N (R ¹² ) S (O) _m- , -N (R ¹² ) S (O) _m N (R ¹³ )-, -N (R ¹² ) C (O)-, -N (R ¹² ) A divalent linking group selected from the group consisting of C (O) N (R ¹³ )-, -N (R ¹² ) C (S)-and -N (R ¹² ) C (O) O-.
However, R ¹² and R ¹³ are independently composed of H, (C ₁ to C ₇ ) alkyl, (C ₁ to C ₇ ) heteroalkyl, aryl and aryl (C ₁ to C ₇ ) alkyl. When selected and, optionally, Y is -N (R ¹² ) S (O) _m -or -N (R ¹² ) S (O) _m N (R ¹³ )-, R ¹² is A covalent bond to Ar or R ² forms a 5- or 6-membered ring that fuses to Ar or R ² , respectively, where the subscript m is an integer of 1-2;
However, R ¹ is OH, and -Y-R ² is -N (R ¹² ) S (O) _m -R ² or -N (R ¹² ) C (O) N (R ¹³ ) -R ² . And when it is attached to the para position with respect to the quaternary carbon attached to Ar, and when R ² is phenyl, benzyl or benzoyl, i) at least one of R ¹² or R ¹³ is other than hydrogen. Yes, and contains an electron-withdrawing substituent, or ii) R ² is amino, acetamide, di (C ₁ to C ₇ ) alkyl amino, (C ₁ to C ₇ ) alkyl amino, halogen, hydroxy, Substituted with a component different from _nitro or ( _C1-7 ) alkyl, or iii) the benzene ring moiety of ^R2 is at least three independent in addition to the Y group or in addition to the bond to Y. Is replaced by the selected group.

In some embodiments, Y is −N (R ¹² ) S (O) _2- and R ¹ is OH.

式Ｉの化合物は、米国特許第６，３１６，５０３号（これは参照によって本明細書中に組み込まれる）によって記載されるように合成することができる。 Thus, compounds of formula I include, but are not limited to, compounds having the structures shown below:

Compounds of formula I can be synthesized as described in US Pat. No. 6,316,503, which is incorporated herein by reference.

（式中、
Ｒ^１はＨである；
Ｘ^１は、結合、Ｃ_１～Ｃ_５アルキル、－Ｃ（Ｏ）－、－Ｃ（＝ＣＲ^８Ｒ^９）－、－Ｏ－、－Ｓ（Ｏ）_ｔ－、－ＮＲ^８－、－ＣＲ^８Ｒ^９－、－ＣＨＲ^２３、－ＣＲ^８（ＣＲ^９）－、－Ｃ（ＣＲ^８）_２－、－ＣＲ_８（ＯＣ（Ｏ）Ｒ^９）－、－Ｃ＝ＮＯＲ^９－、－Ｃ（Ｏ）ＮＲ^８－、－ＣＨ_２Ｏ－、－ＣＨ_２Ｓ－、－ＣＨ_２ＮＲ^８－、－ＯＣＨ_２－、－ＳＣＨ_２－、－ＮＲ^８ＣＨ_２－または

である；
Ｒ^２は、Ｈ、Ｃ_１～Ｃ_６アルキル、Ｃ_２～Ｃ_６アルケニル、Ｃ_２～Ｃ_６アルキニル、Ｃ_３～Ｃ_６シクロアルキル、－ＣＨ_２ＯＨ、Ｃ_７～Ｃ_１１アリールアルキル、フェニル、ナ
フチル、Ｃ_１～Ｃ_３ペルフルオロアルキル、ＣＮ、Ｃ（Ｏ）ＮＨ_２、ＣＯ_２Ｒ^１２、または、Ｃ_１～Ｃ_３アルキル、Ｃ_２～Ｃ_４アルケニル、Ｃ_２～Ｃ_４アルキニル、Ｃ_１～Ｃ_３アルコキシ、Ｃ_１～Ｃ_３ペルフルオロアルキル、ハロゲン、－ＮＯ_２、－ＮＲ^８Ｒ^９、－ＣＮ、－ＯＨ、および、１個～５個のフッ素により置換されるＣ_１～Ｃ_３アルキルから独立して選択される群の１つまたは複数によって独立して置換されるフェニルであり、あるいは、Ｒ^２は、ピリジン、チオフェン、ベンゾイソオキサゾール、ベンゾチオフェン、オキサジアゾール、ピロール、ピラゾール、イミダゾールおよびフランからなる群から選択される複素環であり、この場合、それらのそれぞれが場合により、Ｃ_１～Ｃ_３アルキル、Ｃ_１～Ｃ_３アルコキシ、Ｃ_１～Ｃ_３ペルフルオロアルキル、ハロゲン、－ＮＯ_２、－ＮＲ^８Ｒ^９、－ＣＮ、および、１個～５個のフッ素により置換されるＣ_１～Ｃ_３アルキルから独立して選択される１つ～３つの基により置換される場合がある；
Ｘ^２は結合または－ＣＨ_２－である；
Ｒ^３は、フェニル、ナフチル、あるいは、Ｃ_１～Ｃ_３アルキル、ヒドロキシ、フェニル、アシル、ハロゲン、－ＮＨ_２、－ＣＮ、－ＮＯ_２、Ｃ_１～Ｃ_３アルコキシ、Ｃ_１～Ｃ_３ペルフルオロアルキル、１個～５個のフッ素により置換されるＣ_１～Ｃ_３アルキル、ＮＲ^１４Ｒ^１５、－Ｃ（Ｏ）Ｒ^１０、
－Ｃ（Ｏ）ＮＲ^１０Ｒ^１１、－Ｃ（Ｏ）ＮＲ^１１Ａ、－Ｃ≡ＣＲ^８、－ＣＨ＝ＣＨＲ^８、－ＷＡ、－Ｃ≡ＣＡ、－ＣＨ＝ＣＨＡ、－ＷＹＡ、－ＷＹＮＲ^１１－Ａ、
－ＷＹＲ^１０、－ＷＹ（ＣＨ２）_ｊＡ、－ＷＣＨＲ^１１（ＣＨ２）_ｊＡ、－Ｗ（ＣＨ２）_ｊＡ、－Ｗ（ＣＨ_２）_ｊＲ^１０、－ＣＨＲ^１１Ｗ（ＣＨ_２）_ｊＲ^１０、
－ＣＨＲ^１１Ｗ（ＣＨ_２）_ｊＡ、－ＣＨＲ^１１ＮＲ^１２ＹＡ、－ＣＨＲ^１１ＮＲ^１２ＹＲ^１０、ピロール、－Ｗ（ＣＨ_２）_ｊＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、
－Ｗ（ＣＲ^１８Ｒ^１９）Ａ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－（ＣＨ_２）_ｊＷＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－ＣＨ＝ＣＨＡ（ＣＨ２）_ｋＤ（ＣＨ２）ｐＺ、－Ｃ≡ＣＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－Ｗ（ＣＨ_２）_ｊＣ≡ＣＡ（ＣＨ_２）_ｋＤ（ＣＨ２）_ｐＺおよび－Ｗ（ＣＨ_２）_ｊＺから独立して選択される１つ～４つの基によって独立して置換されるフェニルまたはナフチルであり、
あるいは、Ｒ^３は、ピリミジン、チオフェン、フラン、ベンゾチオフェン、インドール、ベンゾフラン、ベンゾイミダゾール、ベンゾチアゾール、ベンゾオキサゾールおよびキノリンから選択される複素環であり、この場合、それらのそれぞれが場合により、Ｃ_１～Ｃ_３アルキル、Ｃ_１～Ｃ_３アルコキシ、ヒドロキシ、フェニル、アシル、ハロゲン、－ＮＨ_２、－ＣＮ、－ＮＯ_２、Ｃ_１～Ｃ_３ペルフルオロアルキル、１個～５個のフッ素により置換されるＣ_１～Ｃ_３アルキル、－Ｃ（Ｏ）Ｒ^１０、－Ｃ（Ｏ）ＮＲ^１０Ｒ^１１、－Ｃ（Ｏ）ＮＲ^１１Ａ、－Ｃ≡ＣＲ^８、－ＣＨ＝ＣＨＲ^８、－ＷＡ、－Ｃ≡ＣＡ、－ＣＨ＝ＣＨＡ、－ＷＹＡ、－ＷＹＲ^１０、－ＷＹ（ＣＨ_２）_ｊＡ、
－Ｗ（ＣＨ_２）_ｊＡ、－Ｗ（ＣＨ_２）_ｊＲ^１０、－ＣＨＲ^１１Ｗ（ＣＨ_２）_ｊＲ^１０、－ＣＨＲ^１１Ｗ（ＣＨ_２）_ｊＡ、－ＣＨＲ^１１ＮＲ^１２ＹＡ、－ＣＨＲ^１１ＮＲ^１２ＹＲ^１０、
－ＷＣＨＲ^１１（ＣＨ_２）_ｊＡ、－Ｗ（ＣＨ_２）_ｊＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－Ｗ（ＣＲ^１８Ｒ^１９）Ａ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－（ＣＨ_２）_ｊＷＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－ＣＨ＝ＣＨＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－Ｃ≡ＣＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－Ｗ（ＣＨ_２）_ｊＣ≡ＣＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺおよび－Ｗ（ＣＨ_２）_ｊＺ
から独立して選択される１つ～３つの基により置換される場合がある；
Ｗは、結合、－Ｏ－、－Ｓ－、－Ｓ（Ｏ）－、－Ｓ（Ｏ）_２－、－ＮＲ^１１－または－Ｎ（ＣＯＲ^１２）－である；
Ｙは、－ＣＯ－、－Ｓ（Ｏ）^２－、－ＣＯＮＲ^１３、－ＣＯＮＲ^１３ＣＯ－、－ＣＯＮＲ^１３ＳＯ_２－、－Ｃ（ＮＣＮ）－、－ＣＳＮＲ^１３、－Ｃ（ＮＨ）ＮＲ^１３または－Ｃ（Ｏ）Ｏ－である；
ｊは０～３である；
ｋは０～３である；
ｔは０～２である；
Ｄは、結合、－ＣＨ＝ＣＨ－、－Ｃ≡Ｃ－、－Ｃ＝、－Ｃ（Ｏ）－、フェニル、－Ｏ－、－ＮＨ－、－Ｓ－、－ＣＨＲ^１４－、－ＣＲ^１４Ｒ^１５－、－ＯＣＨＲ^１４、－ＯＣＲ^１４Ｒ^１５－または－ＣＨ（ＯＨ）ＣＨ（ＯＨ）－である；
ｐは０～３である；
Ｚは、－ＣＯ_２Ｒ^１１、－ＣＯＮＲ^１０Ｒ^１１、－Ｃ（ＮＲ^１０）ＮＲ^１１Ｒ^１２、－ＣＯＮＨ_２ＮＨ_２、－ＣＮ、－ＣＨ_２ＯＨ、－ＮＲ^１６Ｒ^１７、フェニル、ＣＯＮＨＣＨ（Ｒ^２０）ＣＯＲ^１２、フタルイミド、ピロリジン－２，５ジオン、チアゾリジン－２，４－ジオン、テトラゾリル、ピロール、インドール、オキサゾール、２－チオキソ－１，３－チアゾリニン－４－オン、Ｃ_１～Ｃ_７アミン、Ｃ_３～Ｃ_７環状アミン、または、１個～２個のＯＨ基により置換されるＣ_１～Ｃ_３アルキルであり、但し、前記ピロールは場合により、－ＣＯ_２ＣＨ_３、－ＣＯ_２Ｈ、－ＣＯＣＨ_３、－ＣＯＮＨ_２および－ＣＮからなる群から独立して選択される１つまたは２つの置換基により置換され、
前記Ｃ_１～Ｃ_７アミンは場合により、－ＯＨ、ハロゲン、－ＯＣＨ_３および－Ｃ≡ＣＨからなる群から独立して選択される１つまたは２つの置換基により置換され、
前記フェニルは場合により、ＣＯ_２Ｒ^１１により置換され、かつ、前記Ｃ_３～Ｃ_７環状アミンは場合により、－ＯＨ、－ＣＨ_２ＯＨ、Ｃ_１～Ｃ_３アルキル、－ＣＨ_２ＯＣＨ_３、－ＣＯ_２ＣＨ_３および－ＣＯＮＨ_２からなる群から独立して選択される１つまたは２つの置換基により置換され、かつ、前記オキサゾールは場合により、ＣＨ_２ＣＯ_２Ｒ^１１により置換される；
Ａは、フェニル、ナフチル、テトラヒドロナフチル、インダンまたはビフェニルであり、この場合、それらのそれぞれが場合により、ハロゲン、Ｃ_１～Ｃ_３アルキル、Ｃ_２～Ｃ_４アルケニル、Ｃ_２～Ｃ_４アルキニル、アシル、ヒドロキシ、ハロゲン、－ＣＮ、－ＮＯ_２、－ＣＯ_２Ｒ^１１、－ＣＨ_２ＣＯ_２Ｒ^１１、フェニル、Ｃ_１～Ｃ_３ペルフルオロアルコキシ、Ｃ_１～Ｃ_３ペルフルオロアルキル、－ＮＲ^１０Ｒ^１１、－ＣＨ_２ＮＲ^１０Ｒ^１１、－ＳＲ^１１、１個～５個のフッ素により置換されるＣ_１～Ｃ_６アルキル、１個～２個のＯＨ基により置換されるＣ_１～Ｃ_３アルキル、１個～５個のフッ素により場合により置換されるＣ_１～Ｃ_６アルコキシ、または、１個～２個のＣＦ_３基により場合により置換されるフェノキシから独立して選択される１つ～４つの基により置換される場合がある；あるいは
Ａは、ピロール、ピリジン、ピリジン－Ｎ－オキシド、ピリミジン、ピラゾール、チオフェン、フラン、キノリン、オキサゾール、チアゾール、イミダゾール、イソオキサゾール、インドール、ベンゾ［１，３］－ジオキソール、ベンゾ［１，２，５］－オキソジアゾール、イソクロメン－１－オン、ベンゾチオフェン、ベンゾフラン、２，３－ジ－５ヒドロベンゾ［１，４］－ジオキシン、ビテイニル、キナゾリン－２，４－９［３Ｈ］ジオンおよび３－Ｈ－イソベンゾフラン－１－オンから選択される複素環であり、この場合、それらのそれぞれが場合により、ハロゲン、Ｃ_１～Ｃ_３アルキル、アシル、ヒドロキシ、－ＣＮ、－ＮＯ_２、Ｃ_１～Ｃ_３ペルフルオロアルキル、－ＮＲ^１０Ｒ^１１、－ＣＨ_２ＮＲ^１０Ｒ^１１、－ＳＲ^１１、１個～５個のフッ素により置換されるＣ_１～Ｃ_３アルキル、および、１個～５個のフッ素により場合により置換されるＣ_１～Ｃ_３アルコキシから独立して選択される１つ～３つの基により置換される場合がある；
Ｒ^４、Ｒ^５およびＲ^６はそれぞれが独立して、－Ｈまたは－Ｆである；
Ｒ^７は、Ｃ_１～Ｃ_４アルキル、Ｃ_１～Ｃ_４ペルフルオロアルキル、ハロゲン、－ＮＯ_２、－ＣＮ、フェニル、あるいは、ハロゲン、Ｃ_１～Ｃ_２アルキルおよびＯＨから独立して選択される１つまたは２つの基により置換されるフェニルである；
但し、Ｘ_１Ｒ^２が水素を形成するならば、Ｒ^３は、
（ａ）－Ｗ（ＣＨ_２）_ｊＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－Ｗ（ＣＲ^１８Ｒ^１９）Ａ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－（ＣＨ_２）_ｊＷＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－ＣＨ＝ＣＨＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－Ｃ≡ＣＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺま
たは－Ｗ（ＣＨ_２）_ｊＣ≡ＣＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺによって置換されるフェニル（但し、フェニル成分はさらに場合により、Ｃ_１～Ｃ_２アルキル、Ｃ_１～Ｃ_２ペルフルオロアルキル、ハロゲンおよびＣＮから独立して選択される１つまたは２つの基により置換される）；および
（ｂ）ピリミジン、チオフェンおよびフランから選択される複素環（但し、これらのそれぞれが、－Ｗ（ＣＨ_２）_ｊＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－Ｗ（ＣＲ^１８Ｒ^１９）Ａ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－（ＣＨ２）_ｊＷＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－ＣＨ＝ＣＨＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺ、－Ｃ≡ＣＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺまたは－Ｗ（ＣＨ_２）_ｊＣ≡ＣＡ（ＣＨ_２）_ｋＤ（ＣＨ_２）_ｐＺのうちの１つによって置換される）
から選択される；
それぞれのＲ^８は独立して、－ＨまたはＣ_１～Ｃ_３アルキルである；
それぞれのＲ^９は独立して、－ＨまたはＣ_１～Ｃ_３アルキルである；
それぞれのＲ^１０は独立して、－Ｈ、－ＣＨ、Ｃ_１～Ｃ_３アルコキシ、Ｃ_１～Ｃ_７アルキル、Ｃ_３～Ｃ_７アルケニル、Ｃ_３～Ｃ_７アルキニル、Ｃ_３～Ｃ_７シクロアルキル、－ＣＨ_２ＣＨ_２ＯＣＨ_３、２－メチル－テトラヒドロ－フラン、２－メチル－テトラヒドロ－ピラン、４－メチル－ピペリジン、モルホリン、ピロリジン、あるいは、１個または２個のＣ_１～Ｃ_３アルコキシ基により場合により置換されるフェニルであり、但し、前記Ｃ_１～Ｃ_７アルキルは場合により、Ｃ_１～Ｃ_３アルコキシ、Ｃ_１～Ｃ_３チオアルコキシおよびＣＮから独立して置換される１つ、２つまたは３つの基により置換される；
それぞれのＲ^１１は独立して、－Ｈ、Ｃ_１～Ｃ_３アルキルまたはＲ^２２であり、あるいは、Ｒ^１０およびＲ^１１は、同じ原子に結合するとき、前記原子と一緒になって、
Ｃ_１～Ｃ_３アルキル、ＯＨおよびＣ_１～Ｃ_３アルコキシから独立して選択される１つまたは２つの基により場合により置換される５員～７員の飽和環、あるいは、１個または２個のヘテロ原子を含有し、Ｃ_１～Ｃ_３アルキル、ＯＨおよびＣ_１～Ｃ_３アルコキシから独立して選択される１つまたは２つの基によって場合により置換される５員～７員の環
を形成する：
それぞれのＲ^１２は独立して、－ＨまたはＣ_１～Ｃ_３アルキルである；
それぞれのＲ^１３は独立して、－ＨまたはＣ_１～Ｃ_３アルキルである；
それぞれのＲ^１４およびＲ^１５は独立して、Ｃ_１～Ｃ_７アルキル、Ｃ_３～Ｃ_８シクロアルキル、Ｃ_２～Ｃ_７アルケニル、Ｃ_２～Ｃ_７アルキニル、－ＣＨ、－Ｆ、Ｃ_７～Ｃ_１４アリールアルキルであり、但し、前記アリールアルキルは場合により、ＮＯ_２、Ｃ_１～Ｃ_６アルキル、Ｃ_１～Ｃ_３ペルハロアルキル、ハロゲン、ＣＨ_２ＣＯ_２Ｒ^１１、フェニルおよびＣ_１～Ｃ_３アルコキシから独立して選択される１つ～３つの基により置換され、あるいは、Ｒ^１２およびＲ^１５は、それらが結合する原子と一緒になって、３員～７員の飽和環を形成することができる；
それぞれのＲ^１６およびＲ^１７は独立して、水素、Ｃ_１～Ｃ_３アルキル、Ｃ_１～Ｃ_３アルケニル、Ｃ_１～Ｃ_３アルキニル、フェニル、ベンジルまたはＣ_３～Ｃ_８シクロアルキルであり、但し、前記Ｃ_１～Ｃ_３アルキルは場合により、１つのＯＨ基により置換され、かつ、前記ベンジルは場合により、Ｃ_１～Ｃ_３アルキルおよびＣ_１～Ｃ_３アルコキシから選択される１つ～３つの基により置換され、あるいは、Ｒ^１６およびＲ^１７は、それらが結合する原子と一緒になって、Ｃ_１～Ｃ_３アルキル、－ＯＨ、ＣＨ_２ＯＨ、－ＣＨ_２ＯＣＨ_３、－ＣＯ_２ＣＨ_３および－ＣＯＮＨ_２からなる群から独立して選択される１つまたは２つの置換基により場合により置換される３員～８員の複素環を形成することができる；
それぞれのＲ^１８およびＲ^１９は独立して、Ｃ_１～Ｃ_３アルキルである；
それぞれのＲ^２０は独立して、Ｈ、フェニル、または、天然に存在するアルファアミノ酸の側鎖である；
それぞれのＲ^２２は独立して、ＣＨ_２ＣＯＯＨにより場合により置換されるアリールアルキルである；かつ
それぞれのＲ_２３はフェニルである）
またはその医薬的に許容される塩。 Formula II is provided below:

(During the ceremony,
R ¹ is H;
X ¹ is bonded, C ₁ to C ₅ alkyl, -C (O)-, -C (= CR ⁸ R ⁹ )-, -O-, -S (O) _t- , -NR ^8- , -CR ⁸ R ^9- , -CHR ²³ , -CR ⁸ (CR ⁹ )-, -C (CR ⁸ ) _2- , -CR ₈ (OC (O) R ⁹ )-, -C = NOR ^9- , -C ( O) NR ^8- , -CH ₂ O-, -CH ₂ S-, -CH ₂ NR ^8- , -OCH _2- , -SCH _2- , -NR ⁸ CH ₂ -or

Is;
R ² is H, C ₁ to C ₆ alkyl, C ₂ to C ₆ alkenyl, C ₂ to C ₆ alkynyl, C ₃ to C ₆ cycloalkyl, -CH ₂ OH, C ₇ to C ₁₁ arylalkyl, phenyl, Naftyl, C ₁ to C ₃ perfluoroalkyl, CN, C (O) NH ₂ , CO ₂ R ¹² , or C ₁ to C ₃ alkyl, C ₂ to C ₄ alkenyl, C ₂ to C ₄ alkynyl, C ₁ to From C ₃ alkoxy, C ₁ to C ₃ perfluoroalkyl, halogen, -NO ₂ , -NR ⁸ R ⁹ , -CN, -OH, and C ₁ to C ₃ alkyl substituted with 1 to 5 fluorines. A phenyl that is independently substituted by one or more of the independently selected groups, or R ² is pyridine, thiophene, benzoisoxazole, benzothiophene, oxadiazole, pyrrole, pyrazole, imidazole and It is _a heterocycle selected from the group consisting of furans, in which case _each of them is C1 to C3 alkyl, C1 to C3 alkoxy _, C1 to C3 perfluoroalkyl _, halogen _, -NO ₂ as the case _may be. , -NR ⁸ R ⁹ , -CN, and may be substituted with 1 to 3 groups independently selected from the C ₁ to C ₃ alkyl substituted by 1 to 5 fluorines;
X ² is a bond or -CH _2- ;
R ³ is phenyl, naphthyl, or C ₁ to C ₃ alkyl, hydroxy, phenyl, acyl, halogen, -NH ₂ , -CN, -NO ₂ , C ₁ to C ₃ alkoxy, C ₁ to C ₃ perfluoroalkyl. C ₁ to C ₃ alkyl substituted with 1 to 5 fluorines, NR ¹⁴ R ¹⁵ , -C (O) R ¹⁰ ,
-C (O) NR ¹⁰ R ¹¹ , -C (O) NR ¹¹ A, -C≡CR ⁸ , -CH = CHR ⁸ , -WA, -C≡CA, -CH = CHA, -WYA, -WYNR ¹¹ -A,
-WYR ¹⁰ , -WY (CH2) _j A, -WCHR ¹¹ (CH2) _j A, -W (CH2) _j A, -W (CH ₂ ) _j R ¹⁰ , -CHR ¹¹ W (CH ₂ ) _j R ¹⁰ ,
-CHR ¹¹ W (CH ₂ ) _j A, -CHR ¹¹ NR ¹² YA, -CHR ¹¹ NR ¹² YR ¹⁰ , Pyrrole, -W (CH ₂ ) _j A (CH ₂ ) _k D (CH ₂ ) _p Z,
-W (CR ¹⁸ R ¹⁹ ) A (CH ₂ ) _k D (CH ₂ ) _p Z,-(CH ₂ ) _j WA (CH ₂ ) _k D (CH ₂ ) _p Z, -CH = CHA (CH 2) _k D (CH2) pZ, _-C≡CA ( _CH2 ) _k D ( _CH2 ) pZ, -W ( _CH2 ) _{j C≡CA} ( _CH2 ) kD (CH2) pZ and -W _{( CH2} ) ) _Phenyl or naphthyl independently substituted with one to four groups independently selected from jZ.
Alternatively, R ³ is a heterocycle selected from pyrimidines, thiophenes, furans, benzothiophenes, indols, benzofurans, benzimidazoles, benzothiazoles, benzoxazoles and quinolines, each of which is optionally C ₁ . Substituting with ~ C ₃ alkyl, C ₁ ~ C ₃ alkoxy, hydroxy, phenyl, acyl, halogen, -NH ₂ , -CN, -NO ₂ , C ₁ ~ C ₃ perfluoroalkyl, 1 ~ 5 fluorine C ₁ to C ₃ alkyl, -C (O) R ¹⁰ , -C (O) NR ¹⁰ R ¹¹ , -C (O) NR ¹¹ A, -C≡CR ⁸ , -CH = CHR ⁸ , -WA,- C≡CA, -CH = CHA, -WYA, -WYR ¹⁰ , -WY (CH ₂ ) _j A,
-W (CH ₂ ) _j A, -W (CH ₂ ) _j R ¹⁰ , -CHR ¹¹ W (CH ₂ ) _j R ¹⁰ , -CHR ¹¹ W (CH ₂ ) _j A, -CHR ¹¹ NR ¹² YA,- CHR ¹¹ NR ¹² YR ¹⁰ ,
-WCHR ¹¹ (CH ₂ ) _j A, -W (CH ₂ ) _j A (CH ₂ ) _k D (CH ₂ ) _p Z, -W (CR ¹⁸ R ¹⁹ ) A (CH ₂ ) _k D (CH ₂ ) _p Z,-(CH ₂ ) _j WA (CH ₂ ) _k D (CH ₂ ) _p Z, -CH = CHA (CH ₂ ) _k D (CH ₂ ) _p Z, -C≡CA (CH ₂ ) _k D (CH ₂ ) _p Z, -W (CH ₂ ) _j C≡CA (CH ₂ ) _k D (CH ₂ ) _p Z and -W (CH ₂ ) _j Z
May be replaced by one to three groups selected independently of;
W is a bond, -O-, -S-, -S (O)-, -S (O) _2- , -NR ^11- or -N (COR ¹² )-;
Y is -CO-, -S (O) ^2- , -CONR ¹³ , -CONR ¹³ CO-, -CONR ¹³ SO _2- , -C (NCN)-, -CSNR ¹³ , -C (NH) NR ¹³ Or -C (O) O-;
j is 0 to 3;
k is 0 to 3;
t is 0 to 2;
D is bound, -CH = CH-, -C≡C-, -C =, -C (O)-, phenyl, -O-, -NH-, -S-, -CHR ^14- , -CR ¹⁴ R ^15- , -OCHR ¹⁴ , -OCR ¹⁴ R ^15- or -CH (OH) CH (OH)-;
p is 0 to 3;
Z is -CO ₂ R ¹¹ , -CONR ¹⁰ R ¹¹ , -C (NR ¹⁰ ) NR ¹¹ R ¹² , -CONH ₂ NH ₂ , -CN, -CH ₂ OH, -NR ¹⁶ R ¹⁷ , phenyl, CONHCH ( R ²⁰ ) COR ¹² , phthalimide, pyrrolidine-2,5 dione, thiazolidine-2,4-dione, tetrazolyl, pyrrole, indole, oxazole, 2-thioxo-1,3-thiazolinin-4-one, C ₁ to C ₇ Amines, C ₃ to C ₇ cyclic amines, or C ₁ to C ₃ alkyl substituted with 1 to 2 OH groups, provided that the pyrrole is optionally -CO ₂ CH ₃ , -CO ₂ Substituted with one or two substituents independently selected from the group consisting of H, -COCH ₃ , -CONH ₂ and -CN,
The C ₁ to C ₇ amines are optionally substituted with one or two substituents independently selected from the group consisting of -OH, halogen, -OCH ₃ and -C≡CH.
The phenyl is optionally substituted with CO ₂ R ¹¹ , and the C ₃ to C ₇ cyclic amines are optionally -OH, -CH ₂ OH, C ₁ to C ₃ alkyl, -CH ₂ OCH ₃ , -. Substituted with one or two substituents independently selected from the group consisting of CO ₂ CH ₃ and -CONH ₂ , and the oxazole is optionally substituted with CH ₂ CO ₂ R ¹¹ ;
A is phenyl, naphthyl, tetrahydronaphthyl, indan or biphenyl, each of which is optionally halogen, C1 _- C3 alkyl, _{C2 - C4} alkenyl, _C2 - _C4 alkynyl, acyl. , Hydroxy, Halogen, -CN, -NO ₂ , -CO ₂ R ¹¹ , -CH ₂ CO ₂ R ¹¹ , Phenyl, C ₁ to C ₃ Perfluoroalkryl, C ₁ to C ₃ Perfluoroalkyl, -NR ¹⁰ R ¹¹ , -CH ₂ NR ¹⁰ R ¹¹ , -SR ¹¹ , C ₁ to C ₆ alkyl substituted with 1 to 5 fluorines, C ₁ to C ₃ alkyl substituted with 1 to 2 OH groups, 1 1 to 4 groups independently selected from C ₁ to C ₆ alkoxy optionally substituted with 1 to 5 fluorines, or phenoxy optionally substituted with 1 to 2 CF ₃ groups. Alternatively, A may be substituted with pyrrole, pyridine, pyridine-N-oxide, pyrimidine, pyrazole, thiophene, furan, quinoline, oxazole, thiazole, imidazole, isooxazole, indol, benzo [1,3]-. Dioxol, benzo [1,2,5] -oxodiazol, isochromen-1-one, benzothiophene, benzofuran, 2,3-di-5hydrobenzo [1,4] -dioxin, bitinyl, quinazoline-2,4- It is a heterocycle selected from 9 [3H] dione and ₃ -H-isobenzofuran- ₁ -one, in which case each of them is optionally halogen, C1 to C3 alkyl, acyl, hydroxy, -CN. , -NO ₂ , C ₁ to C ₃ perfluoroalkyl, -NR ¹⁰ R ¹¹ , -CH ₂ NR ¹⁰ R ¹¹ , -SR ¹¹ , 1 to 5 fluorine substituted C ₁ to C ₃ alkyl, and May be substituted with 1 to _{3 groups independently selected from the C 1-3 alkoxy} optionally substituted with 1 to 5 fluorides;
R ⁴ , R ⁵ and R ⁶ are independently -H or -F;
R ⁷ is independently selected from C ₁ to C ₄ alkyl, C ₁ to C ₄ perfluoroalkyl, halogen, -NO ₂ , -CN, phenyl, or halogen, C ₁ to C ₂ alkyl and OH 1 Phenyl substituted by one or two groups;
However, if X ₁ R ² forms hydrogen, then R ³ is
(A) -W (CH ₂ ) _j A (CH ₂ ) _k D (CH ₂ ) _p Z, -W (CR ¹⁸ R ¹⁹ ) A (CH ₂ ) _k D (CH ₂ ) _p Z,-(CH ₂ ) ) _J WA (CH ₂ ) _k D (CH ₂ ) _p Z, -CH = CHA (CH ₂ ) _k D (CH ₂ ) _p Z, -C≡CA (CH ₂ ) _k D (CH ₂ ) _p Z or -W (CH ₂ ) _j C≡CA (CH ₂ ) _k D (CH ₂ ) phenyl substituted with _p Z (provided that the phenyl component is further C ₁ to C ₂ alkyl, C ₁ to C ₂ perfluoro depending on the case. Substituted by one or two groups independently selected from alkyl, halogen and CN); and (b) heterocycles selected from pyrimidines, thiophenes and furans, each of which is -W ( CH ₂ ) _j A (CH ₂ ) _k D (CH ₂ ) _p Z, -W (CR ¹⁸ R ¹⁹ ) A (CH ₂ ) _k D (CH ₂ ) _p Z,-(CH 2) _j WA (CH ₂ ) _k D (CH ₂ ) _p Z, -CH = CHA (CH ₂ ) _k D (CH ₂ ) _p Z, -C≡CA (CH ₂ ) _k D (CH ₂ ) _p Z or -W (CH ₂ ) _{j C≡CA} (CH ₂ ) _k D (CH ₂ ) replaced by one of pZ)
Selected from;
Each R ⁸ is independently -H or C ₁ to C ₃ alkyl;
Each R ⁹ is independently -H or C ₁ to C ₃ alkyl;
Each R ¹⁰ is independently -H, -CH, C ₁ to C ₃ alkoxy, C ₁ to C ₇ alkyl, C ₃ to C ₇ alkenyl, C ₃ to C ₇ alkynyl, C ₃ to C ₇ cycloalkyl. , -CH ₂ CH ₂ OCH ₃ , 2-methyl-tetrahydro-furan, 2-methyl-tetrahydro-pyran, 4-methyl-piperidine, morpholine, pyrrolidine, or one or two C ₁ to C ₃ alkoxy groups. _{The C1} to _C7 alkyl is optionally substituted with _one , _two , independently of C1 to C3 alkoxy _, C1 to C3 thioalkoxy and CN. Substituted by one or three groups;
Each R ¹¹ is independently -H _{, C 1-3 alkyl} or R ²² , or R ¹⁰ and R ¹¹ together with said atom when bonded to the same atom,
A 5- to 7-membered saturated ring, or one or two, optionally substituted with one or two groups independently selected from C ₁ to C ₃ alkyl, OH and C ₁ to C ₃ alkoxy. Heteroatoms are contained and form a 5- to 7-membered ring optionally substituted by one or two groups independently selected from C ₁ to C ₃ alkyl, OH and C ₁ to C ₃ alkoxy. do:
Each R ¹² is independently -H or C ₁ to C ₃ alkyl;
Each R ¹³ is independently -H or C ₁ to C ₃ alkyl;
Each of R ¹⁴ and R ¹⁵ is independently C ₁ to C ₇ alkyl, C ₃ to C ₈ cycloalkyl, C ₂ to C ₇ alkoxy, C ₂ to C ₇ alkynyl, -CH, -F, C ₇ to. C ₁₄ arylalkyl, provided that the arylalkyl is NO ₂ , C ₁ to C ₆ alkyl, C ₁ to C ₃ perhaloalkyl, halogen, CH ₂ CO ₂ R ¹¹ , phenyl and C ₁ to C ₃ as the case may be. Substituted by one to three groups independently selected from alkoxy, or R ¹² and R ¹⁵ together with the atoms to which they are attached form a 3- to 7-membered saturated ring. Can be;
Each of R ¹⁶ and R ¹⁷ is independently hydrogen, C ₁ to C ₃ alkyl, C ₁ to C ₃ alkoxy, C ₁ to C ₃ alkynyl, phenyl, benzyl or C ₃ to C ₈ cycloalkyl, provided that , The C ₁ to C ₃ alkyl are optionally substituted with one OH group, and the benzyl is optionally one to three selected from C ₁ to C ₃ alkyl and C ₁ to C ₃ alkoxy. Substituted by a group, or R ¹⁶ and R ¹⁷ together with the atom to which they are attached, C ₁ to C ₃ alkyl, -OH, CH ₂ OH, -CH ₂ OCH ₃ , -CO ₂ CH ₃ And -A 3- to 8-membered heterocycle optionally substituted with one or two substituents selected independently of the group consisting of CONH ₂ can be formed;
_Each of R ¹⁸ and R ¹⁹ is independently C1 to _C3 alkyl;
Each ^R20 is independently a side chain of H, phenyl, or a naturally occurring alpha amino acid;
Each R ²² is independently an arylalkyl optionally substituted with CH ₂ COOH; and each R ₂₃ is phenyl).
Or its pharmaceutically acceptable salt.

式ＩＩＩが下記において提供される：

（式中、
Ｘは、水素、Ｃ_１～Ｃ_８アルキル、ハロ、－ＯＲ^１０、－ＮＲ^１０Ｒ^１１、ニトロ、シアノ、－ＣＯＯＲ^１０または－ＣＯＲ^１０から選択される；
Ｚは、ＣＨ、ＣＲ^３またはＮであり、但し、ＺがＣＨまたはＣＲ^３であるときには、ｋは０～４であり、かつ、ｔは０または１であり、また、ＺがＮであるときには、ｋは０～３であり、かつ、ｔは０である；
Ｙは、－Ｏ－、－Ｓ－、－Ｎ（Ｒ^１２）－および－Ｃ（Ｒ^４）（Ｒ^５）－から選択される；
Ｗ^１は、Ｃ_１～Ｃ_６アルキル、Ｃ_０～Ｃ_６アルキル、Ｃ_３～Ｃ_６シクロアルキル、アリールおよびＨｅｔから選択され、但し、前記Ｃ_１～Ｃ_８アルキル、Ｃ_３～Ｃ_８シクロアルキル、ＡｒおよびＨｅｔは場合により非置換であるか、あるいは、ハロ、シアノ、ニトロ、Ｃ_１～Ｃ_６アルキル、Ｃ_３～Ｃ_６アルケニル、Ｃ_３～Ｃ_６アルキニル、－Ｃ_０～Ｃ_６アルキル－ＣＯ_２Ｒ^１２、－Ｃ_０～Ｃ_６アルキル－Ｃ（Ｏ）ＳＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＣＯＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＳＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＯＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＳＯ_３Ｈ、－Ｃ_０～Ｃ_６アルキル－ＳＯ_２ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＳＯ_２Ｒ^１２、－Ｃ_０～Ｃ_６アルキル－ＳＯＲ^１５、－Ｃ_０～Ｃ_６アルキルＯＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＯＣ（Ｏ）ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＯＣ（Ｏ）ＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｃ（Ｏ）ＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｃ（Ｏ）ＮＲ^１３Ｒ^１４および－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３ＣＯＲ^１５から独立して選択される１つまたは複数の基により置換され、この場合、前記Ｃ_１～Ｃ_６アルキルは場合により非置換であるか、あるいは、１つまたは複数のハロ置換基によって置換される；
Ｗ^２は、Ｈ、ハロ、Ｃ_１～Ｃ_６アルキル、Ｃ_２～Ｃ_６アルケニル、Ｃ_２～Ｃ_６アルキニル、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＳＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＯＲ^１２、－Ｃ_０～Ｃ_６アルキルＣＯ_２Ｒ^１２、－Ｃ_０～Ｃ_６アルキル－Ｃ（Ｏ）ＳＲ^１２、－Ｃ_０～Ｃ_６アルキルＣＯＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキルＯＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＯＣＯＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３ＣＯＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３ＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－Ｈｅｔ、－Ｃ_０～Ｃ_６アルキル－Ａｒおよび－Ｃ_０～Ｃ_６アルキル－Ｃ_３～Ｃ_７シクロアルキルから選択され、但し、前記Ｃ_１～Ｃ_６アルキルは場合により非置換であるか、あるいは、１つまたは複数のハロ置換基によって置換され、かつ、前記－Ｃ_０～Ｃ_６アルキル－Ｈｅｔ、－Ｃ_０～Ｃ_６アルキル－Ａｒおよび－Ｃ_０～Ｃ_６アルキル－Ｃ_３～Ｃ_７シクロアルキルのＣ_３～Ｃ_７シクロアルキル部分、Ａｒ部分およびＨｅｔ部分は場合により非置換であるか、あるいは、ハロ、シアノ、ニトロ、Ｃ_１～Ｃ_６アルキル、Ｃ_３～Ｃ_６アルケニル、Ｃ_３～Ｃ_６アルキニル、－Ｃ_０
～Ｃ_６アルキル－ＣＯ２Ｒ^１２、－Ｃ_０～Ｃ_６アルキル－Ｃ（Ｏ）ＳＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＣＯＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＳＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＯＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＳＯ_３Ｈ、－Ｃ_０～Ｃ_６アルキル－ＳＯ_２ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＳＯ_２Ｒ^１２、－Ｃ_０～Ｃ_６アルキル－ＳＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＯＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＯＣ（Ｏ）ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＯＣ（Ｏ）ＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｃ（Ｏ）ＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｃ（Ｏ）ＮＲ^１３Ｒ^１４および－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３ＣＯＲ^１５から独立して選択される１つまたは複数の基により置換され、この場合、前記Ｃ_１～Ｃ_６アルキルは場合により非置換であるか、あるいは、１つまたは複数のハロ置換基によって置換される；
Ｗ^３は、Ｈ、ハロ、Ｃ_１～Ｃ_６アルキル、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキルＳＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＯＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＣＯ_２Ｒ^１２、－Ｃ_０～Ｃ_６アルキル－Ｃ（Ｏ）ＳＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＣＯＮＲ^１３Ｒ^１４、
－Ｃ_０～Ｃ_６アルキル－ＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＯＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＯＣＯＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキルＮＲ^１３ＣＯＮＲ^１３Ｒ^１４、
－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３ＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－Ｈｅｔ、－Ｃ_１～Ｃ_６アルキル－Ａｒおよび－Ｃ_１～Ｃ_６アルキル－Ｃ_３～Ｃ_７シクロアルキルからなる群から選択され、
但し、前記Ｃ_１～Ｃ_６アルキルは場合により非置換であるか、あるいは、１つまたは複数のハロ置換基によって置換される；
Ｑは、Ｃ_３～Ｃ_８シクロアルキル、ＡｒおよびＨｅｔから選択され、但し、前記Ｃ_３～Ｃ_８シクロアルキル、ＡｒおよびＨｅｔは場合により非置換であるか、あるいは、ハロ、シアノ、ニトロ、Ｃ_１～Ｃ_６アルキル、Ｃ_３～Ｃ_６アルケニル、Ｃ_３～Ｃ_６アルキニル、－Ｃ_０～Ｃ_６アルキルＣＯ_２Ｒ^１２、－Ｃ_０～Ｃ_６アルキル－Ｃ（Ｏ）ＳＲ^１２、－Ｃ_０～Ｃ_６アルキルＣＯＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキルＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＳＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＯＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＳＯ_３Ｈ、－Ｃ_０～Ｃ_６アルキル－ＳＯ_２ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＳＯ_２Ｒ^１２、－Ｃ_０～Ｃ_６アルキル－ＳＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＯＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＯＣ（Ｏ）ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＯＣ（Ｏ）ＯＲ^１５、－Ｃ_０～Ｃ_６アルキルＮＲ^１３Ｃ（Ｏ）ＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｃ（Ｏ）ＮＲ^１３Ｒ^１４および－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３ＣＯＲ^１５から独立して選択される１つまたは複数の基により置換され、この場合、前記Ｃ_１Ｃ_６アルキルは場合により非置換であるか、あるいは、１つまたは複数のハロ置換基によって置換される；
ｐは０～８である；
ｎは２～８である；
ｍは０または１である；
ｑは０または１である；
ｔは０または１である；
それぞれのＲ^１およびＲ^２は独立して、Ｈ、ハロ、Ｃ_１～Ｃ_６アルキル、Ｃ_３～Ｃ_６アルケニル、Ｃ_３～Ｃ_６アルキニル、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＯＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＳＲ^１２、－Ｃ_１～Ｃ_６アルキル－Ｈｅｔ、－Ｃ_１～Ｃ_６アルキル－Ａｒおよび－Ｃ_１～Ｃ_６アルキル－Ｃ_３～Ｃ_７シクロアルキルから選択されるか、あるいは、Ｒ^１およびＲ^２は、それらが結合する炭素と一緒になって、３員～５員の炭素環式環または複素環式環を形成し、この場合、前記複素環式環は、Ｎ、ＯおよびＳから選択される１つまたは複数のヘテロ原子を含有し、但し、前記Ｃ_１～Ｃ_６アルキルのいずれもが場合により非置換であるか、あるいは、１つまたは複数のハロ
置換基によって置換される；
それぞれのＲ^３は同じまたは異なり、独立して、ハロ、シアノ、ニトロ、Ｃ_１～Ｃ_６アルキル、Ｃ_３～Ｃ_６アルケニル、Ｃ_３～Ｃ_６アルキニル、－Ｃ_０～Ｃ_６アルキル－Ａｒ、－Ｃ_０～Ｃ_６アルキル－Ｈｅｔ、－Ｃ_０～Ｃ_６アルキル－Ｃ_３～Ｃ_７シクロアルキル、－Ｃ_０～Ｃ_６アルキル－ＣＯ２Ｒ^１２、－Ｃ_０～Ｃ_６アルキル－Ｃ（Ｏ）ＳＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＣＯＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＳＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＯＲ^１２、－Ｃ_０～Ｃ_６アルキル－ＳＯ_３Ｈ、－Ｃ_０～Ｃ_６アルキルＳＯ_２ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＳＯ_２Ｒ^１２、－Ｃ_０～Ｃ_６アルキルＳＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＯＣＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＯＣ（Ｏ）ＮＲ^１３Ｒ^１４、－Ｃ_０～Ｃ_６アルキル－ＯＣ（Ｏ）ＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｃ（Ｏ）ＯＲ^１５、－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３Ｃ（Ｏ）ＮＲ^１３Ｒ^１４および－Ｃ_０～Ｃ_６アルキル－ＮＲ^１３ＣＯＲ^１５から選択され、但し、前記Ｃ_１～Ｃ_６アルキルは場合により非置換であるか、あるいは、１つまたは複数のハロ置換基によって置換される；
それぞれのＲ^４およびＲ^５は独立して、Ｈ、ハロ、Ｃ_１～Ｃ_６アルキル、－Ｃ_０～Ｃ_６アルキル－Ｈｅｔ、－Ｃ_０～Ｃ_６アルキル－Ａｒおよび－Ｃ_０～Ｃ_６アルキル－Ｃ_３～Ｃ_７シクロアルキルから選択される；
Ｒ^６およびＲ^７はそれぞれが独立して、Ｈ、ハロ、Ｃ_１～Ｃ_６アルキル、－Ｃ_０～Ｃ_６アルキル－Ｈｅｔ、－Ｃ_０～Ｃ_６アルキル－Ａｒおよび－Ｃ_０～Ｃ_６アルキル－Ｃ_３～Ｃ_７シクロアルキルから選択される；
Ｒ^８およびＲ^９はそれぞれが独立して、Ｈ、ハロ、Ｃ_１～Ｃ_６アルキル、－Ｃ_０～Ｃ_６アルキル－Ｈｅｔ、－Ｃ_０～Ｃ_６アルキル－Ａｒおよび－Ｃ_０～Ｃ_６アルキル－Ｃ_３～Ｃ_７シクロアルキルから選択される；
Ｒ^１０およびＲ^１１はそれぞれが独立して、Ｈ、Ｃ_１～Ｃ_１２アルキル、Ｃ_３～Ｃ_１２アルケニル、Ｃ_３～Ｃ_１２アルキニル、
－Ｃ_０～Ｃ_８アルキル－Ａｒ、－Ｃ_０～Ｃ_８アルキル－Ｈｅｔ、－Ｃ_０～Ｃ_８アルキル－Ｃ_３～Ｃ_７シクロアルキル、－Ｃ_０～Ｃ_８アルキル－Ｏ－Ａｒ、－Ｃ_０～Ｃ_８アルキル－Ｏ－Ｈｅｔ、
－Ｃ_０～Ｃ_８アルキル－Ｏ－Ｃ_３～Ｃ_７シクロアルキル、－Ｃ_０～Ｃ_８アルキル－Ｓ（Ｏ）_ｘ－Ｃ_０～Ｃ_６アルキル、－Ｃ_０～Ｃ_８アルキル－Ｓ（Ｏ）_ｘ－Ａｒ、－Ｃ_０～Ｃ_８アルキル－Ｓ（Ｏ）_ｘ－Ｈｅｔ、－Ｃ_０～Ｃ_８アルキル－Ｓ（Ｏ）_ｘ－Ｃ_３～Ｃ_７シクロアルキル、－Ｃ_０～Ｃ_８アルキル－ＮＨ－Ａｒ、－Ｃ_０～Ｃ_８アルキル－ＮＨ－Ｈｅｔ、－Ｃ_０～Ｃ_８アルキル－ＮＨ－Ｃ_３～Ｃ_７シクロアルキル、－Ｃ_０～Ｃ_８アルキル－Ｎ（Ｃ_１～Ｃ_４アルキル）－Ａｒ、－Ｃ_０～Ｃ_８アルキル－Ｎ（Ｃ_１～Ｃ_４アルキル）－Ｈｅｔ、－Ｃ_０～Ｃ_８アルキル－Ｎ（Ｃ_１～Ｃ_４アルキル－Ｃ_３～Ｃ_７シクロアルキル、－Ｃ_０～Ｃ_８アルキル－Ａｒ、－Ｃ_０～Ｃ_８アルキル－Ｈｅｔおよび－Ｃ_０～Ｃ_８アルキル－Ｃ_３～Ｃ_７シクロアルキルから選択され、この場合、ｘは、０、１または２であり、
あるいは、Ｒ^１０およびＲ^１１は、それらが結合する窒素と一緒になって、Ｎ、ＯおよびＳから選択される１つまたは複数のさらなるヘテロ原子を場合により含有する４員～７員の複素環式環を形成し、但し、前記Ｃ_１～Ｃ_１２アルキル、Ｃ_３～Ｃ_１２アルケニルまたはＣ_３～Ｃ_１２アルキニルは場合により、ハロ、－ＯＨ、－ＳＨ、－ＮＨ_２、－ＮＨ（非置換Ｃ_１～Ｃ_６アルキル）、－Ｎ（非置換Ｃ_１～Ｃ_６アルキル）（非置換Ｃ_１～Ｃ_６アルキル）、非置換の－ＯＣ_１～Ｃ_６アルキル、－ＣＯ_２Ｈ、－ＣＯ_２（非置換Ｃ_１～Ｃ_６アルキル）、－ＣＯＮＨ_２、－ＣＯＮＨ（非置換Ｃ_１～Ｃ_６アルキル）、－ＣＯＮ（非置換Ｃ_１～Ｃ_６アルキル）（非置換Ｃ_１～Ｃ_６アルキル）、－ＳＯ_３Ｈ、－ＳＯ_２ＮＨ_２、－ＳＯ_２ＮＨ（非置換Ｃ_１～Ｃ_６アルキル）および－ＳＯ_２Ｎ（非置換Ｃ_１～Ｃ_６アルキル）（非置換Ｃ_１～Ｃ_６アルキル）の群から独立して選択される置換基の１つまたは複数によって置換される；
Ｒ^１２は、Ｈ、Ｃ_１～Ｃ_６アルキル、Ｃ_３～Ｃ_６アルケニル、Ｃ_３～Ｃ_６アルキニル、－Ｃ_０～Ｃ_６アルキル－Ａｒ、－Ｃ_０～Ｃ_６アルキル－Ｈｅｔおよび－Ｃ_０～Ｃ_６アルキ
ル－Ｃ_３～Ｃ_７シクロアルキルから選択される；
それぞれのＲ^１３およびそれぞれのＲ^１４は独立して、Ｈ、Ｃ_１～Ｃ_６アルキル、Ｃ_３～Ｃ_６アルケニル、Ｃ_３～Ｃ_６アルキニル、－Ｃ_０～Ｃ_６アルキル－Ａｒ、－Ｃ_０～Ｃ_６アルキル－Ｈｅｔおよび－Ｃ_０～Ｃ_６アルキル－Ｃ_３～Ｃ_７シクロアルキルから選択され、あるいは、Ｒ^１３およびＲ^１４は、それらが結合する窒素と一緒になって、Ｎ、ＯおよびＳから選択される１つまたは複数のさらなるヘテロ原子を場合により含有する４員～７員の複素環式環を形成する；
Ｒ^１５は、Ｃ_１～Ｃ_６アルキル、Ｃ_３～Ｃ_６アルケニル、Ｃ_３～Ｃ_６アルキニル、－Ｃ_０～Ｃ_６アルキル－Ａｒ、－Ｃ_０～Ｃ_６アルキル－Ｈｅｔおよび－Ｃ_０～Ｃ_６アルキル－Ｃ_３～Ｃ_７シクロアルキルから選択される）
またはその医薬的に許容される塩。 Compounds of formula II can be synthesized as described in US Pat. No. 7,576,215, which is incorporated herein by reference. The compound of formula II can be any of compounds 26 to 32 or a pharmaceutically acceptable salt thereof.

Equation III is provided below:

(During the ceremony,
^X is selected from hydrogen, C1 to C8 alkyl, halo, -OR ₁₀ , -NR ₁₀ R ¹¹ , nitro, cyano, -COOR ¹⁰ or -COR ^{10 ;}
Z is CH, CR ³ or N, where k is 0-4 and t is 0 or 1 when Z is CH or CR ³ , and Z is N. , K is 0 to 3 and t is 0;
Y is selected from -O-, -S-, -N (R ¹² )-and -C (R ⁴ ) (R ⁵ )-;
W ¹ is selected from C ₁ to C ₆ alkyl, C ₀ to C ₆ alkyl, C ₃ to C ₆ cycloalkyl, aryl and Het, provided that C ₁ to C ₈ alkyl and C ₃ to C ₈ cycloalkyl are selected. , Ar and Het are optionally unsubstituted or halo, cyano, nitro, C ₁ to C ₆ alkyl, C ₃ to C ₆ alkenyl, C ₃ to C ₆ alkynyl, -C ₀ to C ₆ alkyl- CO ₂ R ¹² , -C ₀ to C ₆ Alkyne-C (O) SR ¹² , -C ₀ to C ₆ Alkyne-CONR ¹³ R ¹⁴ , -C ₀ to C ₆ Alkyne-COR ¹⁵ , -C ₀ to C ₆ Alkyne-NR ¹³ R ¹⁴ , -C ₀ to C ₆ Alkyne-SR ¹² , -C ₀ to C ₆ Alkyne-OR ¹² , -C ₀ to C ₆ Alkyne-SO _3H , -C ₀ to C ₆ Alkyne-SO ₂ NR ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl-SO ₂ R ¹² , -C ₀ to C ₆ alkyl-SOR ¹⁵ , -C ₀ to C ₆ alkyl OCOR ¹⁵ , -C ₀ to C ₆ alkyl-OC ( O) NR ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl-OC (O) OR ¹⁵ , -C ₀ to C ₆ alkyl-NR ¹³ C (O) OR ¹⁵ , -C ₀ to C ₆ alkyl-NR ¹³ C (O) Substituted by one or more groups independently selected from NR ¹³ R ¹⁴ and -C ₀ to C ₆ alkyl-NR ¹³ COR ¹⁵ , in which case the C ₁ to C ₆ alkyl are optionally. Unsubstituted or substituted with one or more halo substituents;
W ² is H, halo, C ₁ to C ₆ alkyl, C ₂ to C ₆ alkenyl, C ₂ to C ₆ alkynyl, -C ₀ to C ₆ alkyl-NR ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl- SR ¹² , -C ₀ to C ₆ Alkyne-OR ¹² , -C ₀ to C ₆ Alkyne CO ₂ R ¹² , -C ₀ to C ₆ Alkyne-C (O) SR ¹² , -C ₀ to C ₆ Alkyne CONR ¹³ R ¹⁴ , -C ₀ to C ₆ Alkyne-COR ¹⁵ , -C ₀ to C ₆ Alkyne OCOR ¹⁵ , -C ₀ to C ₆ Alkyne-OCONR ¹³ R ¹⁴ , -C ₀ to C ₆ Alkyne-NR ¹³ CONR ¹³ R ¹⁴ , -C ₀ to C ₆ Alkyne-NR ¹³ COR ¹⁵ , -C ₀ to C ₆ Alkyne-Het, -C ₀ to C ₆ Alkyne-Ar and -C ₀ to C ₆ Alkyne-C ₃ to C ₇ Cycloalkyl C1 to C6 alkyl are optionally unsubstituted or substituted with one or more halo substituents and the _-C0 to _C6 alkyl _{- Het} ,-. The C ₃ to C ₇ cycloalkyl moieties, Ar moieties and Het moieties of C ₀ to C ₆ alkyl-Ar and -C ₀ to C ₆ alkyl-C ₃ to C ₇ cycloalkyl are optionally unsubstituted or substituted. Halo, cyano, nitro, C ₁ to C ₆ alkyl, C ₃ to C ₆ alkenyl, C ₃ to C ₆ alkynyl, -C ₀
-C ₆ alkyl-CO2R ¹² , -C ₀ to C ₆ alkyl-C (O) SR ¹² , -C ₀ to C ₆ alkyl-CONR ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl-COR ¹⁵ , -C ₀ -C ₆ alkyl-NR ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl-SR ¹² , -C ₀ to C ₆ alkyl-OR ¹² , -C ₀ to C ₆ alkyl-SO ₃ H, -C ₀ to C ₆ Alkyl-SO ₂ NR ¹³ R ¹⁴ , -C ₀ to C ₆ Alkyl-SO ₂ R ¹² , -C ₀ to C ₆ Alkyl-SOR ¹⁵ , -C ₀ to C ₆ Alkyl-OCOR ¹⁵ , -C ₀ to C ₆ Alkyl-OC (O) NR ¹³ R ¹⁴ , -C ₀ to C ₆ Alkyl-OC (O) OR ¹⁵ , -C ₀ to C ₆ Alkyl-NR ¹³ C (O) OR ¹⁵ , -C ₀ to C ₆ Alkyl -NR ¹³ C (O) NR ¹³ R ¹⁴ and -C ₀ to C ₆ alkyl-NR ¹³ COR ¹⁵ substituted with one or more groups independently selected, in this case C ₁ to C ₆ described above. The alkyl is optionally unsubstituted or substituted with one or more halo substituents;
W ³ is H, halo, C ₁ to C ₆ alkyl, -C ₀ to C ₆ alkyl-NR ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl SR ¹² , -C ₀ to C ₆ alkyl-OR ¹² ,- C ₀ to C ₆ alkyl-CO ₂ R ¹² , -C ₀ to C ₆ alkyl-C (O) SR ¹² , -C ₀ to C ₆ alkyl-CONR ¹³ R ¹⁴ ,
-C ₀ to C ₆ alkyl-COR ^{15, -C 0 to C 6 alkyl-OCOR 15} _{, -C 0 to C 6} ^alkyl _{- OCONR} ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl NR ¹³ CONR ¹³ R ¹⁴ ,
Consists of -C ₀ to C ₆ alkyl-NR ¹³ COR ¹⁵ , -C ₀ to C ₆ alkyl-Het, -C ₁ to C ₆ alkyl-Ar and -C ₁ to C ₆ alkyl-C ₃ to C ₇ cycloalkyl Selected from the group,
However, the C ₁ to C ₆ alkyl are optionally unsubstituted or substituted with one or more halo substituents;
Q is selected from C ₃ to C ₈ cycloalkyl, Ar and Het, provided that the C ₃ to C ₈ cycloalkyl, Ar and Het are optionally unsubstituted or halo, cyano, nitro, C. ₁ to C ₆ alkyl, C ₃ to C ₆ alkenyl, C ₃ to C ₆ alkynyl, -C ₀ to C ₆ alkyl CO ₂ R ¹² , -C ₀ to C ₆ alkyl-C (O) SR ¹² , -C ₀ -C ₆ Alkyne CONR ¹³ R ¹⁴ , -C ₀ to C ₆ Alkyne-COR ¹⁵ , -C ₀ to C ₆ Alkyne NR ¹³ R ¹⁴ , -C ₀ to C ₆ Alkyne-SR ¹² , -C ₀ to C ₆ Alkyne -OR ¹² , -C ₀ to C ₆ alkyl-SO ₃ H, -C ₀ to C ₆ alkyl-SO ₂ NR ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl-SO ₂ R ¹² , -C ₀ to C ₆ Alkyne-SOR ¹⁵ , -C ₀ to C ₆ Alkyne-OCOR ¹⁵ , -C ₀ to C ₆ Alkyne-OC (O) NR ¹³ R ¹⁴ , -C ₀ to C ₆ Alkyne-OC (O) OR ¹⁵ , -C Select independently from ₀ to C ₆ alkyl NR ¹³ C (O) OR ¹⁵ , -C ₀ to C ₆ alkyl-NR ¹³ C (O) NR ¹³ R ¹⁴ and -C ₀ to C ₆ alkyl-NR ¹³ COR ¹⁵ . Substituted by one or more groups to be substituted, in which case the C ₁ C ₆ alkyl is optionally unsubstituted or substituted by one or more halo substituents;
p is 0-8;
n is 2-8;
m is 0 or 1;
q is 0 or 1;
t is 0 or 1;
R ¹ and R ² are independently H, halo, C ₁ to C ₆ alkyl, C ₃ to C ₆ alkenyl, C ₃ to C ₆ alkynyl, -C ₀ to C ₆ alkyl-NR ¹³ R ¹⁴ , respectively. -C ₀ to C ₆ alkyl-OR ¹² , -C ₀ to C ₆ alkyl-SR ¹² , -C ₁ to C ₆ alkyl-Het, -C ₁ to C ₆ alkyl-Ar and -C ₁ to C ₆ alkyl- Selected from C ₃ to C ₇ cycloalkyl, or R ¹ and R ² together with the carbon to which they are attached form a 3- to 5-membered carbocyclic or heterocyclic ring. However, in this case, the heterocyclic ring contains one or more heteroatoms selected from N, O and S, provided that any of the C ₁ to C ₆ alkyl is optionally unsubstituted. Is or is substituted with one or more halo substituents;
Each R ³ is the same or different and independently, halo, cyano, nitro, C ₁ to C ₆ alkyl, C ₃ to C ₆ alkenyl, C ₃ to C ₆ alkynyl, -C ₀ to C ₆ alkyl-Ar, -C ₀ to C ₆ alkyl-Het, -C ₀ to C ₆ alkyl-C ₃ to C ₇ cycloalkyl, -C ₀ to C ₆ alkyl-CO2R ¹² , -C ₀ to C ₆ alkyl-C (O) SR ¹² , -C ₀ to C ₆ alkyl-CONR ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl-COR ¹⁵ , -C ₀ to C ₆ alkyl-NR ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl-SR ¹² , -C ₀ to C ₆ alkyl-OR ¹² , -C ₀ to C ₆ alkyl-SO ₃ H, -C ₀ to C ₆ alkyl SO ₂ NR ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl-SO ₂ R ¹² , -C ₀ to C ₆ alkyl SOR ¹⁵ , -C ₀ to C ₆ alkyl-OCOR ¹⁵ , -C ₀ to C ₆ alkyl-OC (O) NR ¹³ R ¹⁴ , -C ₀ to C ₆ alkyl-OC (O) OR ¹⁵ , -C ₀ to C ₆ alkyl-NR ¹³ C (O) OR ¹⁵ , -C ₀ to C ₆ alkyl-NR ¹³ C (O) NR ¹³ R ¹⁴ and -C ₀ to C ₆ alkyl-NR ¹³ COR ¹⁵ are selected, provided that the C1 to C6 _alkyls are optionally unsubstituted or substituted with _one or more halo substituents;
R ⁴ and R ⁵ are independently H, halo, C ₁ to C ₆ alkyl, -C ₀ to C ₆ alkyl-Het, -C ₀ to C ₆ alkyl-Ar and -C ₀ to C ₆ alkyl, respectively. -Selected from C ₃ to C ₇ cycloalkyl;
R ⁶ and R ⁷ are independently H, halo, C ₁ to C ₆ alkyl, -C ₀ to C ₆ alkyl-Het, -C ₀ to C ₆ alkyl-Ar and -C ₀ to C ₆ alkyl, respectively. -Selected from C ₃ to C ₇ cycloalkyl;
R ⁸ and R ⁹ are independently H, halo, C ₁ to C ₆ alkyl, -C ₀ to C ₆ alkyl-Het, -C ₀ to C ₆ alkyl-Ar and -C ₀ to C ₆ alkyl, respectively. -Selected from C ₃ to C ₇ cycloalkyl;
R ¹⁰ and R ¹¹ are independently H, C ₁ to C ₁₂ alkyl, C ₃ to C ₁₂ alkenyl, C ₃ to C ₁₂ alkynyl, respectively.
-C ₀ to C ₈ alkyl-Ar, -C ₀ to C ₈ alkyl-Het, -C ₀ to C ₈ alkyl-C ₃ to C ₇ cycloalkyl, -C ₀ to C ₈ alkyl-O-Ar, -C _{0 -} C8 alkyl-O-Het,
-C ₀ to C ₈ alkyl-OC ₃ to C ₇ cycloalkyl, -C ₀ to C ₈ alkyl-S (O) _x -C ₀ to C ₆ alkyl, -C ₀ to C ₈ alkyl-S (O) ) _X -Ar, -C ₀ to C ₈ alkyl-S (O) _x -Het, -C ₀ to C ₈ alkyl-S (O) _x -C ₃ to C ₇ cycloalkyl, -C ₀ to C ₈ alkyl -NH-Ar, -C ₀ to C ₈ alkyl-NH-Het, -C ₀ to C ₈ alkyl-NH-C ₃ to C ₇ cycloalkyl, -C ₀ to C ₈ alkyl-N (C ₁ to C ₄ ) Alkyl) -Ar, -C ₀ to C ₈ alkyl-N (C ₁ to C ₄ alkyl) -Het, -C ₀ to C ₈ alkyl-N (C ₁ to C ₄ alkyl-C ₃ to C ₇ cycloalkyl, It is selected from -C ₀ to C ₈ alkyl-Ar, -C ₀ to C ₈ alkyl-Het and -C ₀ to C ₈ alkyl-C ₃ to C ₇ cycloalkyl, where x is 0, 1 or 2 And
Alternatively, R ¹⁰ and R ¹¹ are 4- to 7-membered heterocycles, optionally containing one or more additional heteroatoms selected from N, O and S, together with the nitrogen to which they are attached. Forming a ring, provided that the C ₁ to C ₁₂ alkyl, C ₃ to C ₁₂ alkenyl or C ₃ to C ₁₂ alkynyl may optionally be halo, -OH, -SH, -NH ₂ , -NH (unsubstituted). C ₁ to C ₆ alkyl), -N (unsubstituted C ₁ to C ₆ alkyl) (unsubstituted C ₁ to C ₆ alkyl), unsubstituted -OC ₁ to C ₆ alkyl, -CO ₂ H, -CO ₂ (Unsubstituted C ₁ to C ₆ alkyl), -CONH ₂ , -CONH (unsubstituted C ₁ to C ₆ alkyl), -CON (unsubstituted C ₁ to C ₆ alkyl) (unsubstituted C ₁ to C ₆ alkyl) , -SO ₃ H, -SO ₂ NH ₂ , -SO ₂ NH (unsubstituted C ₁ to C ₆ alkyl) and -SO ₂ N (unsubstituted C ₁ to C ₆ alkyl) (unsubstituted C ₁ to C ₆ alkyl). ) Is replaced by one or more of the substituents selected independently;
R ¹² includes H, C ₁ to C ₆ alkyl, C ₃ to C ₆ alkenyl, C ₃ to C ₆ alkynyl, -C ₀ to C ₆ alkyl-Ar, -C ₀ to C ₆ alkyl-Het and -C ₀ . ~ C ₆ Alkyne-C ₃ ~ C ₇ Cyclo Alkyne;
Each R ¹³ and each R ¹⁴ independently have H, C ₁ to C ₆ alkyl, C ₃ to C ₆ alkenyl, C ₃ to C ₆ alkynyl, -C ₀ to C ₆ alkyl-Ar, -C ₀ . Selected from ~ C ₆ Alkyne-Het and -C ₀ ~ C ₆ Alkyne-C ₃ ~ C ₇ Cycloalkyl, or R ¹³ and R ¹⁴ together with the nitrogen to which they bind, N, O and Form a 4- to 7-membered heterocyclic ring optionally containing one or more additional heteroatoms selected from S;
R ¹⁵ is C ₁ to C ₆ alkyl, C ₃ to C ₆ alkenyl, C ₃ to C ₆ alkynyl, -C ₀ to C ₆ alkyl-Ar, -C ₀ to C ₆ alkyl-Het and -C ₀ to C. (Selected from ₆ alkyl-C ₃ to C ₇ cycloalkyl)
Or its pharmaceutically acceptable salt.

In some embodiments, X is hydrogen, p is 0, t is 0, Z is CH, and Y is —O—.

In a further embodiment, X is hydrogen, p is 0, t is 0, Z is CH, and Y is —O—, W ¹ and W ² are phenyl, and W. ³ is hydrogen, q is 1, and R ⁸ and R ⁹ are hydrogen.

In other embodiments, X is hydrogen, p is 0, t is 0, Z is CH, Y is —O—, and W ¹ and W ² are phenyl. W ³ is hydrogen, q is 1, and R ⁸ and R ⁹ are hydrogen, and Q is Ar.

式ＩＩＩの化合物は、米国特許第７，３６５，０８５号および同第７，５６０，５８６号（これらは参照によって本明細書中に組み込まれる）に記載されるように合成することができる。
式ＩＶが下記に示される：

またはその医薬的に許容される塩、但し、式ＩＶにおいて、
Ｊ^１１は－Ｎ＝であり、かつ、Ｊ^２１は－ＣＲ^３００－であり、または、Ｊ^１１は－ＣＲ^２００－であり、かつ、Ｊ２１は＝Ｎ－である；
Ｒ^００は、Ｇ^１、Ｇ^２１またはＲ^Ｎである；
Ｒ^２００は、Ｇ^１、Ｇ^２１またはＲ^Ｃである；
Ｒ^３００およびＲ^４００は独立して、Ｒ^ＣまたはＱであり、但し、Ｒ^３００、Ｒ^４００およびＲ^５００のうちのただ１つだけがＱである；
Ｑは、Ｃ_３～６シクロアルキル、ヘテロアリールまたはヘテロシクリルであり、それぞれが場合により、１つ～４つのＲ^Ｑにより置換されるか、あるいは、Ｑは－Ｘ－Ｙ－Ｚであり、但し、それぞれのＲ^Ｑが独立して、アリールオキシ、アラルキルオキシ、アリールオキシアルキル、アリールＣ_０～Ｃ_６アルキルカルボキシ、Ｃ（Ｒ^１１０）＝Ｃ（Ｒ^１１０）－ＣＯＯＨ、オキソ、＝Ｓ、－Ｚ、－Ｙ’－Ｚまたは－Ｘ－Ｙ－Ｚであり、それぞれのＲ^Ｑは場合により、１つ～４つのＲ^８０により置換される；
Ｒ^５００は、Ｇ^１、Ｇ^２１、ＱまたはＲ^Ｃであり、但し、この場合、Ｒ^００、Ｒ^２００およびＲ^５００のうちのただ１つがＧ^１であり、かつ、Ｒ^００、Ｎ＝およびＲ^５００のうちのただ１つがＧ^２１である；
Ｇ^２１は－Ｊ^０－Ｋ^０であり、式中、Ｊ^０およびＫ^０は独立して、アリールまたはヘテロアリールであり、それぞれが場合により、１つ～４つのＲ^Ｋ基により置換され、それぞれのＲ^Ｋが独立して、水素、ハロゲン、ＣＲ^１１０＝ＣＲ^１１０ＣＯＯＲ^１１０、ニトロ、－Ｚ、－Ｙ－Ｚまたは－Ｘ－Ｙ－Ｚである；
Ｇ^１は－Ｌ^１０－Ｒであり、但し、Ｌ^１０は、結合、Ｌ^５０、Ｌ^６０、－Ｌ^５０－Ｌ^６０－Ｌ^５０－または－Ｌ^６０－Ｌ^５０－Ｌ^５０－であり、これらの式において、
それぞれのＬ^５０が独立して、－［Ｃ（Ｒ^１５０）_２］_ｍ－であり、
それぞれのＬ^６０が独立して、－ＣＳ－、－ＣＯ－、－ＳＯ_２－、－Ｏ－、－ＣＯＮ（Ｒ^１１０）－、－ＣＯＮＲ^１１０Ｎ（Ｒ^１１０）－、－Ｃ（＝ＮＲ^１１０）－、－Ｃ（ＮＯＲ^１１）－、－Ｃ（＝Ｎ－Ｎ（Ｒ^１１０）_２）－、－Ｃ_３～Ｃ_８シクロアルキル－または－ヘテロシクリル－であり、前記シクロアルキルまたはヘテロシクリルは場合により、１つ～４つのＲ^１４０基により置換され、あるいは、それぞれのＬ^６０が独立して、Ｃ_２～Ｃ_６アリジイルであり、前記アリジイル鎖は場合により、－Ｃ（Ｒ^１００）_２－、－Ｃ（Ｒ^１１０）_２Ｃ（Ｒ^１１０）_ｚ－、－Ｃ（Ｒ^１１）Ｃ（Ｒ^１１０）－、－Ｃ（Ｒ^１１０）_２Ｏ－、－Ｃ（Ｒ^１１０）_ｚＮＲ^１１０－、－Ｃ Ｃ－、－Ｏ－、－Ｓ－、－Ｎ（ＲＯ）ＣＯ－、－Ｎ（Ｒ^１００）ＣＯ_２－、－ＣＯＮ（Ｒ^１１０）－、－ＣＯ－、－ＣＯ_２－、－ＯＣ（＝Ｏ）－、－ＯＣ（＝Ｏ）Ｎ（Ｒ^１００）－、－ＳＯ_２－、－Ｎ（Ｒ^１００）ＳＯ_２－または－ＳＯ_２Ｎ（Ｒ^１００）によって中断され、
Ｒが、アリール、ヘテロシクリル、ヘテロアリールまたは－（Ｃ_３～Ｃ_６）シクロアルキルであり、但し、Ｒは場合により、１つ～４つのＲ^Ａにより置換され、この場合、それぞれのＲ^Ａが独立して、ハロゲン、ニトロ、ヘテロシクリル、Ｃ_１～Ｃ_６アルキル、Ｃ_２～Ｃ_６アルケニル、Ｃ_２～Ｃ_６アルキニル、Ｃ_３～Ｃ_８シクロアルキル、（Ｃ_３～Ｃ_８シ
クロアルキル）－Ｃ_１～Ｃ_６アルキル－、（Ｃ_３～Ｃ_８シクロアルケニル）－Ｃ_１～Ｃ_６アルキル－、（Ｃ_３～Ｃ_８シクロアルキル）－Ｃ_１～Ｃ_６アルケニル－、アリールアルキル、アリールオキシ、アリールＣ_１～６アルコキシ、Ｃ_１～Ｃ_６ハロアルキル、ＳＯ_２Ｒ^１１０、ＯＲ^１１０、ＳＲ^１１０、Ｎ_３、ＳＯＲ^１１０、ＣＯＲ^１１０、ＳＯ_２Ｎ（Ｒ^１１０）_２、ＳＯ_２ＮＲ^１１０ＣＯＲ^１１０、Ｃ≡Ｎ、Ｃ（Ｏ）ＯＲ^１１０、ＣＯＮ（Ｒ^１１０）_２、－ＣＯＮ（Ｒ^１１０）ＯＲ^１１０、ＯＣＯＮ（Ｒ^１１０）_２、－ＮＲ^１１０ＣＯＲ^１１０、ＮＲ^１１０ＣＯＮ（Ｒ^１１０）_２、ＮＲ^１１０ＣＯＯＲ^１１０、－Ｃ（＝Ｎ－ＯＨ）Ｒ^１１０、－Ｃ（＝Ｓ）Ｎ（Ｒ^１１０）_２、
－Ｓ（＝Ｏ）Ｎ（Ｒ^１１０）_２、－Ｓ（＝Ｏ）ＯＲ^１１０、－Ｎ（Ｒ^１１０）Ｓ（＝Ｏ）_２Ｒ^１１０、－Ｃ（＝Ｏ）Ｎ（Ｒ^１１０）Ｎ（Ｒ^１１０）_２、－ＯＣ（＝Ｏ）－Ｒ^１１０、－ＯＣ（＝Ｏ）－ＯＲ^１１０またはＮ（Ｒ^１１）_２であり、
かつ、それぞれのＲ^Ａが場合により、独立して－ハロゲン、－Ｃ_１～Ｃ_６アルキル、アリールオキシ、Ｃ_０～６アルキルＳＯ_２Ｒ^１１０、Ｃ_０～６アルキルＣＯＯＲ^１１０、Ｃ_１～６アルコキシアリール、Ｃ_１～Ｃ_６ハロアルキル、
－ＳＯ_２Ｒ^１１０、－ＯＲ^１１０、－ＳＲ^１１０、－Ｎ_３、－ＳＯ_２Ｒ^１１０、－ＣＯＲ^１１０、－ＳＯ_２Ｎ（Ｒ^１１０）_２、－ＳＯ_２ＮＲ^１１０ＣＯＲ^１１０、－Ｃ≡Ｎ、－Ｃ（Ｏ）ＯＲ^１１０、
－ＣＯＮ（Ｒ^１１０）_２、－ＣＯＮ（Ｒ^１１０）ＯＲ^１１０、－ＯＣＯＮ（Ｒ^１１０）_２、－ＮＲ^１１０ＣＯＲ^１１０、－ＮＲ^１１０ＣＯＮ（Ｒ^１１０）_２、－ＮＲ^１１０ＣＯＯＲ^１１０または－Ｎ（Ｒ^１１０）_２である１つ～４つの基により置換される；
Ｒ^Ｎは－Ｌ^３１－Ｒ^６０であり、但し、Ｌ^３１は、結合、－Ｘ^３（ＣＨ_ｚ）_ｎ－Ｘ^３－、－（ＣＨ_２）_ｍ－Ｘ３－（ＣＨ_２）_ｎ－または－（ＣＨ_２）_１＋ｗ，－Ｙ^３－（ＣＨ_２）_ｗ－であり、これらの式において、それぞれのｗが独立して、０～５であり、かつ、それぞれのＸ^３が独立して、結合、－Ｃ（Ｒ^１１０）_２－、－Ｃ（Ｒ^１１０）_２Ｃ（Ｒ^１１０）_２－、－Ｃ（Ｒ^１１０）＝Ｃ（Ｒ^１１０）－、－Ｃ≡Ｃ－、－ＣＯ－、－ＣＳ－、－ＣＯＮＲ^１００－、－Ｃ（＝Ｎ）（Ｒ^１００）－、－Ｃ（＝Ｎ－ＯＲ^１１０）－、－Ｃ［＝Ｎ－Ｎ（Ｒ^１１０）_２］、－ＣＯ_２－、－ＳＯ_２－または－ＳＯ_２Ｎ（Ｒ^１１０）－であり、かつ
Ｙ^３が、－Ｏ－、－Ｓ－、－ＮＲ^７０－、－Ｎ（Ｒ^１００）ＣＯ－、－Ｎ（Ｒ^１１０）ＣＯ２－、－ＯＣＯ－、－ＯＣ（＝Ｏ）Ｎ（Ｒ^１００）－、－ＮＲ^１００ＣＯＮＲ^１００－、－Ｎ（Ｒ^１１０）ＳＯ_２－または－ＮＲ^１００ＣＳＮＲ^１００－であり、
あるいは、Ｌ^３１はＣ_２～Ｃ_６アリジイル鎖であり、但し、前記アリジイル鎖は場合により、－Ｃ（Ｒ^１１０）_２－、
－Ｃ（Ｒ^１１０）_２Ｃ（Ｒ^１１０）_２－、－Ｃ（Ｒ^１１０）＝Ｃ（Ｒ^１１０）－、－Ｃ（Ｒ^１１０）_２Ｏ－、－Ｃ（Ｒ^１１０）_２ＮＲ^１１０－、－Ｃ≡Ｃ－、－Ｏ－、－Ｓ－、－Ｎ（Ｒ^１００）ＣＯ－、
－Ｎ（Ｒ^１００）ＣＯ_２－、－ＣＯＮ（Ｒ^１００）－、－ＣＯ－、－ＣＯ_２－、－Ｏ（Ｃ＝Ｏ）－、－Ｏ（Ｃ＝Ｏ）Ｎ（Ｒ^１１０）－、－ＳＯ_２－、－Ｎ（Ｒ^１００）ＳＯ_２－または－ＳＯ_２Ｎ（Ｒ^１００）－によって中断され、
Ｒ^６０が、Ｃ_１～Ｃ_６アルキル、Ｃ_１～Ｃ_６ハロアルキル、アリール、Ｃ_３～Ｃ_８シクロアルキル、ヘテロアリール、ヘテロシクリル、－ＣＮ、
－Ｃ（＝Ｏ）Ｒ^１１０、－Ｃ（＝Ｏ）ＯＲ^１１０、－Ｃ（＝Ｏ）Ｎ（Ｒ^１１０）_２、－Ｎ（Ｒ^１１０）_２、－ＳＯ_２Ｒ^１１０、－Ｓ（＝Ｏ）_２Ｎ（Ｒ^１１０）_２、－Ｃ（＝Ｏ）Ｎ（Ｒ^１１０）Ｎ（Ｒ^１１０）_２または－Ｃ（＝Ｏ）Ｎ（Ｒ^１１）（ＯＲ^１１０）であり、但し、前記アリール、ヘテロアリール、シクロアルキルまたはヘテロシクリルは場合により、１つ～４つのＲ^６０ａにより置換され、この場合、
それぞれのＲ^６０ａが独立して、－Ｚ、－Ｙ’－Ｚまたは－Ｘ－Ｙ－Ｚである；
それぞれのＲ^Ｃは独立して、－Ｌ^３０－Ｒ^７０であり、但し、
それぞれのＬ^３０が独立して、結合または－（ＣＨ_２）_ｍ－Ｖ^１０－（ＣＨ_２）_ｎ－であり、式中、
Ｖ^１０は、－Ｃ（Ｒ^１１０）_２－、－Ｃ（Ｒ^１１０）_２Ｃ（Ｒ^１１０）_２、－Ｃ（Ｒ^１１０）＝Ｃ（Ｒ^１１０）－、－Ｃ（Ｒ^１１０）_２Ｏ－、－Ｃ（Ｒ^１１０）_２ＮＲ^１１０－、－Ｃ≡Ｃ－、－Ｏ－、
－Ｓ－、－ＮＲ^１０－、－Ｎ（Ｒ^１００）ＣＯ－、－Ｎ（Ｒ^１００）ＣＯ２－、－ＯＣＯ－、－ＣＯ－、－ＣＳ－、－ＣＯＮＲ^１００－、－Ｃ（＝Ｎ－Ｒ^１１０）－、－Ｃ（＝Ｎ－ＯＲ^１１０）－、
－Ｃ［＝Ｎ－Ｎ（Ｒ^１１０）_２］、－ＣＯ_２－、－ＯＣ（＝Ｏ）－、－ＯＣ（＝Ｏ）Ｎ（Ｒ^１００）－、ＳＯ_２－、－Ｎ（Ｒ^１００）ＳＯ_２－、－ＳＯ_２Ｎ（Ｒ^１００）－、－ＮＲ^１００ＣＯＮＲ^１００－、－ＮＲ^１００ＣＳＮＲ^１００－、Ｃ_３～Ｃ_６シクロアルキルまたはＣ_３～Ｃ_６シクロハロアルキルであり、
あるいは、それぞれのＬ^３０が独立して、Ｃ_２～Ｃ_６アリジイルであり、但し、前記アリジイル鎖は場合により、－Ｃ（Ｒ^１１０）_２－、－Ｃ（Ｒ^１１０）_２Ｃ（Ｒ^１１０）_２－、－Ｃ（Ｒ^１１０）Ｃ（Ｒ^１１０）－、－Ｃ（Ｒ^１１０）_２Ｏ－、－Ｃ（Ｒ^１１０）_２ＮＲ^１１０－、－Ｃ≡Ｃ－、－Ｏ－、－Ｓ－、－Ｎ（Ｒ^１００）ＣＯ－、
－Ｎ（Ｒ^１００）ＣＯ_２－、－ＮＲ^１１０－、－ＣＯＮ（Ｒ^１００）－、－ＣＯ－、－ＣＯ_２－、－Ｏ（Ｃ＝Ｏ）－、－Ｏ（Ｃ＝Ｏ）Ｎ（Ｒ^１００）－、－ＳＯ_２－、－Ｎ（Ｒ^１００）ＳＯ_２－または－ＳＯ_２Ｎ（Ｒ^１００）－によって中断され、
それぞれのＲ^７０が独立して、水素、ハロゲン、ニトロ、アリール、ヘテロアリール、ヘテロシクリル、－Ｚ、－Ｙ－Ｚまたは－Ｘ－ＹＺであり、
この場合、前記アリール、ヘテロアリールおよびヘテロシクリルはそれぞれが場合により、１つ～４つのＲ^７０ａにより置換され、但し、それぞれのＲ^７０ａが独立し、アリールオキシ、アラルキルオキシ、アリールオキシアルキル、アリールＣ_０～Ｃ_６アルキルカルボキシ、Ｃ（Ｒ^１１０）＝Ｃ（Ｒ^１１０）ＣＯＯＨ、オキソ、－Ｚ、－Ｙ’－Ｚまたは－Ｘ－Ｙ－Ｚであり、かつ、それぞれのＲ^７０ａが場合により、１つ～４つのＲ^８０により置換され、但し、それぞれのＲ^８０が独立して、ハロゲン、Ｃ_１～Ｃ_６アルキル、Ｃ_１～Ｃ_６アルコキシ、Ｃ_１～Ｃ_８ハロアルキル、Ｃ_１～Ｃ_８ハロアルキル（ＯＲ^１１０）、Ｃ_０～Ｃ_６アルキルＯＲ^１１０、Ｃ_０～Ｃ_６アルキルＣＯＮ（Ｒ^１１０）_２、Ｃ_０～Ｃ_６アルキルＣＯＲ^１１０、Ｃ_０～Ｃ_６アルキルＣＯＯＲ^１１０またはＣ_０～Ｃ_６アルキルＳＯ_２Ｒ^１１０である；
それぞれのＲ^１００は独立して、－Ｒ^１１０、－Ｃ（＝Ｏ）Ｒ^１１０、－ＣＯ_２Ｒ^１１０または－ＳＯ_２Ｒ^１１０である；
それぞれのＲ^１１０は独立して、－水素、－Ｃ_１～Ｃ_６アルキル、Ｃ_２～Ｃ_６アルケニル、－Ｃ_１～Ｃ_６アルキニル、－Ｃ_１～Ｃ_６ハロアルキルまたは－Ｎ（Ｒ^１２）_２であり、但し、Ｒ^１１０のいずれもが場合により、Ｒ^１２０の１つ～４つの基により置換される：
それぞれのＲ^１２０は独立して、ハロゲン、シアノ、ニトロ、オキソ、－Ｂ（ＯＲ^１３０）、Ｃ_０～Ｃ_６アルキルＮ（Ｒ^１３）_２、Ｃ_１～Ｃ_６ハロアルキル、Ｃ_１～Ｃ_６アルキル、Ｃ_１～Ｃ_６アルコキシ、（Ｃ_０～Ｃ_６アルキル）Ｃ＝Ｏ（ＯＲ^１３０）、Ｃ_０～Ｃ_６アルキルＯＲ^１３０、Ｃ_０～Ｃ_６アルキルＣＯＲ^１３０、
Ｃ_０～Ｃ_６アルキルＳＯ_２Ｒ^１３０、Ｃ_０～Ｃ_６アルキルＣＯＮ（Ｒ^１３）_２、Ｃ_０～Ｃ_６アルキルＣＯＮＲ^１３０ＯＲ^１３０、Ｃ_０～Ｃ_６アルキルＳＯ_２Ｎ（Ｒ^１３０）_２、Ｃ_０～Ｃ_６アルキルＳＲ^１３０、Ｃ_０～Ｃ_６ハロアルキルＯＲ^１３０、Ｃ_０～Ｃ_６アルキルＣＮ、－Ｃ_０～Ｃ_６アルキＮ（Ｒ^１３）_２、－ＮＲ^１３ＳＯ_２Ｒ^１３または－ＯＣ_０～６アルキルＣＯＯＲ^１３０である；
それぞれのＲ^１３０は独立して、水素、Ｃ_１～Ｃ_６アルキル、Ｃ_２～Ｃ_６アルケニルまたはＣ_２～Ｃ_６アルキニルである；
それぞれのＲ^１４０は独立して、Ｃ_１～Ｃ_６アルキル、Ｃ_１～Ｃ_６アルコキシ、ハロゲン、Ｃ_１～Ｃ_６ハロアルキル、Ｃ_０～Ｃ_６アルキルＣＯＮ（Ｒ^１１０）_ｏ、Ｃ_０～Ｃ_６アルキルＣＯＮＲ^１１０Ｒ^１０、Ｃ_０～Ｃ_６アルキルＯＲ^１１０またはＣ_０～Ｃ_６アルキルＣＯＯＲ^１１０である；かつ
それぞれのＲ^１５０は独立して、水素、ハロゲン、ＯＲ^１３０、（Ｃ_１～Ｃ_６）アルキルまたは（Ｃ_１～Ｃ_６）ハロアルキルであり、但し、
それぞれのアルキルは場合により、それぞれが独立してハロゲン、シアノ、ニトロ、アジド、ＯＲ^１３０、Ｃ（Ｏ）Ｒ^１３０、Ｃ（Ｏ）ＯＲ^１３Ｃ（Ｏ）Ｎ（Ｒ^１３０）_２、Ｎ（Ｒ^１３０）_２、Ｎ（Ｒ^１３０）Ｃ（Ｏ）Ｒ^１３０、Ｎ（Ｒ^１３０）Ｓ（Ｏ）_２Ｒ^１３０、－ＯＣ（Ｏ）ＯＲ^１３０、ＯＣ（Ｏ）Ｎ（Ｒ^１３０）_２、Ｎ（Ｒ^１３０）Ｃ（Ｏ）ＯＲ^１３０、Ｎ（Ｒ^１３０）Ｃ（Ｏ）Ｎ（Ｒ^１３０）、ＳＲ^１３０、Ｓ（Ｏ）Ｒ^１３０、Ｓ（Ｏ）_２Ｒ’またはＳ（Ｏ）_２Ｎ（Ｒ^１３０）_２である少なくとも１つの基により置換され、あるいは、（同じ原子または異なる原子に結合する）２つのＲ^１５０は一緒になって、Ｃ３～Ｃ６シクロアルキルを形成することができる；
それぞれのＸは独立して、－Ｏ－、－Ｓ－または－Ｎ（Ｒ^１００）－である；
それぞれのＹは独立して、－［Ｃ（Ｒ^１５０）_２］_ｐ－または－Ｃ_２～Ｃ_６アルケニルであり、但し、ｐは、１、２、３、４、５または６である；
それぞれのＹ’は独立して、－［Ｃ（Ｒ^１５０）_２］_ｐ－、－Ｃ_２～Ｃ_６アルケニルＣ_３～Ｃ_８シクロアルキルまたはヘテロシクリルであり、但し、前記シクロアルキルまたはヘテロシクリルは場合により、１つ～３つのＺ基により置換される：
それぞれのＺは独立して、－Ｈ、ハロゲン、－ＯＲ^１１０、－ＳＲ^１１０、－Ｃ（＝Ｏ）Ｒ^１１０、－Ｃ（＝Ｏ）ＯＲ^１１０、－Ｃ（＝Ｏ）Ｎ（Ｒ^１１０）_２、
－Ｎ（Ｒ^１００）_２、－Ｎ_３、－ＮＯ_２、－Ｃ（＝Ｎ－ＯＨ）Ｒ^１１０、－Ｃ（＝Ｓ）Ｎ（Ｒ^１１０）_２、－ＣＮ、－Ｓ（＝Ｏ）Ｒ^１１０、－Ｓ（＝Ｏ）Ｎ（Ｒ^１１０）_２、－Ｓ（＝Ｏ）ＯＲ^１１０、
－Ｓ（＝Ｏ）_２Ｒ^１１０、Ｓ（＝Ｏ）_２Ｎ（Ｒ^１１０）_２、－ＮＲ^１１０ＣＯＲ^１１０、－Ｎ（Ｒ^１１０）Ｃ（＝Ｏ）Ｎ（Ｒ^１１０）_２、－Ｎ（Ｒ^１１０）ＣＯＯＲ^１１０、－Ｎ（Ｒ^１１０）Ｓ（＝Ｏ）_２Ｒ^１１０、－Ｃ（＝Ｏ）Ｎ（Ｒ^１１０）Ｎ（Ｒ^１１０）_２、－Ｃ（＝Ｏ）Ｎ（Ｒ^１１０）（ＯＲ^１１０）、－ＯＣ（＝Ｏ）－Ｒ^１１０、－ＯＣ（＝Ｏ）－ＯＲ^１１０または－ＯＣ（＝Ｏ）－Ｎ（Ｒ^１１０）_２である；かつ
それぞれのｍおよびｎは独立して、０、１、２、３、４、５または６である。 Thus, compounds of formula III include, but are not limited to, compounds having the structures shown below, GW3965 [2] and SB742881 [25]:

Compounds of formula III can be synthesized as described in US Pat. Nos. 7,365,085 and 7,560,586, which are incorporated herein by reference.
Equation IV is shown below:

Or its pharmaceutically acceptable salt, provided that in formula IV,
J ¹¹ is -N = and J ²¹ is -CR ^300- , or J ¹¹ is -CR ^200- and J 21 is = N-;
^{R 00} is G ¹ , G ²¹ or RN;
^R200 is ^G1 , ^G21 or ^RC ;
R ³⁰⁰ and R ⁴⁰⁰ are independently ^RC or Q, except that only one of R ³⁰⁰ , R ⁴⁰⁰ and R ⁵⁰⁰ is Q;
Q is C _3-6 cycloalkyl, heteroaryl or heterocyclyl, each optionally substituted with 1 to 4 RQs, or ^Q is -XYZ, provided that. Each RQ is independently aryloxy, aralkyloxy, aryloxyalkyl, aryl C ₀ to C ₆ alkyl carboxy, C ( ^{R 110} ) = C (R ¹¹⁰ ) -COOH, oxo, = S, -Z, -Y'-Z or ^-XYZ , each RQ is optionally replaced by one to four ^R80s ;
R ⁵⁰⁰ is G ¹ , G ²¹ , Q or ^RC , provided that in this case only one of R ⁰⁰ , R ²⁰⁰ and R ⁵⁰⁰ is G ¹ and R ⁰⁰ , N = and R. Only one of the ⁵⁰⁰ is ^G21 ;
G ²¹ is −J ⁰ −K ⁰ , where J ⁰ and K ⁰ are independently aryl or heteroaryl, each optionally substituted with 1 to 4 ^RK groups, respectively. ^RKs are independently hydrogen, halogen, CR ¹¹⁰ = CR ¹¹⁰ COOR ¹¹⁰ , nitro, -Z, -YZ or -XYZ;
G ¹ is -L ¹⁰ -R, where L ¹⁰ is binding, L ⁵⁰ , L ⁶⁰ , -L ⁵⁰ -L ⁶⁰ -L ⁵⁰ -or -L ⁶⁰ -L ⁵⁰ -L ^50- . In the formula of
Each L ⁵⁰ is independently-[C (R ¹⁵⁰ ) ₂ ] _m- ,
Each L ⁶⁰ independently has -CS-, -CO-, -SO _2- , -O-, -CON (R ¹¹⁰ )-, -CONR ¹¹⁰ N (R ¹¹⁰ )-, -C (= NR ¹¹⁰ ). )-, -C (NOR ¹¹ )-, -C (= NN (R ¹¹⁰ ) ₂ )-, -C ₃ to C ₈ cycloalkyl- or -heterocyclyl-, the cycloalkyl or heterocyclyl as the case may be. Substituted by one to four R ¹⁴⁰ groups, or each L ⁶⁰ is independently C ₂ to C ₆ aligyl, said aligyl chain is optionally -C (R ¹⁰⁰ ) ₂ -,-. C (R ¹¹⁰ ) ₂ C (R ¹¹⁰ ) _z- , -C (R ¹¹ ) C (R ¹¹⁰ )-, -C (R ¹¹⁰ ) ₂ O-, -C (R ¹¹⁰ ) _z NR ^110- , -C C-, -O-, -S-, -N (RO) CO-, -N (R ¹⁰⁰ ) CO _2- , -CON (R ¹¹⁰ )-, -CO-, -CO _2- , -OC (= Interrupted by O)-, -OC (= O) N (R ¹⁰⁰ )-, -SO _2- , -N (R ¹⁰⁰ ) SO _2- or -SO ₂ N (R ¹⁰⁰ ),
R is aryl, heterocyclyl, heteroaryl or-(C ₃ to C ₆ ) cycloalkyl, where ^R is optionally substituted with 1 to 4 RAs, where each ^RA is independent. Halogen, nitro, heterocyclyl, C ₁ to C ₆ alkyl, C ₂ to C ₆ alkoxy, C ₂ to C ₆ alkynyl, C ₃ to C ₈ cycloalkyl, (C ₃ to C ₈ cycloalkyl) -C ₁ ~ C ₆ alkyl-, (C ₃ to C ₈ cycloalkoxy) -C ₁ to C ₆ alkyl-, (C ₃ to C ₈ cycloalkyl) -C ₁ to C ₆ alkoxy-, aryl alkyl, aryl oxy, aryl C _{1 to 6} Alkoxy, C ₁ to C ₆ Haloalkyl, SO ₂ R ¹¹⁰ , OR ¹¹⁰ , SR ¹¹⁰ , N ₃ , SOR ¹¹⁰ , COR ¹¹⁰ , SO ₂ N (R ¹¹⁰ ) ₂ , SO ₂ NR ¹¹⁰ COR ¹¹⁰ , C≡ N, C (O) OR ¹¹⁰ , CON (R ¹¹⁰ ) ₂ , -CON (R ¹¹⁰ ) OR ¹¹⁰ , OCON (R ¹¹⁰ ) ₂ , -NR ¹¹⁰ COR ¹¹⁰ , NR ¹¹⁰ CON (R ¹¹⁰ ) ₂ , NR ¹¹⁰ COOR ¹¹⁰ , -C (= N-OH) R ¹¹⁰ , -C (= S) N (R ¹¹⁰ ) ₂ ,
-S (= O) N (R ¹¹⁰ ) ₂ , -S (= O) OR ¹¹⁰ , -N (R ¹¹⁰ ) S (= O) ₂ R ¹¹⁰ , -C (= O) N (R ¹¹⁰ ) N ( R ¹¹⁰ ) ₂ , -OC (= O) -R ¹¹⁰ , -OC (= O) -OR ¹¹⁰ or N (R ¹¹ ) ₂ .
In addition, each ^RA may be independently -halogen, -C ₁ to C ₆ alkyl, aryloxy, C _{0 to 6} alkyl SO ₂ R ¹¹⁰ , C _{0 to 6} alkyl COOR ¹¹⁰ , C _{1 to 6} alkoxy. Aryl, C ₁ to C ₆ haloalkyl,
-SO ₂ R ¹¹⁰ , -OR ¹¹⁰ , -SR ¹¹⁰ , -N ₃ , -SO ₂ R ¹¹⁰ , -COR ¹¹⁰ , -SO ₂ N (R ¹¹⁰ ) ₂ , -SO ₂ NR ¹¹⁰ COR ¹¹⁰ , -C≡N , -C (O) OR ¹¹⁰ ,
-CON (R ¹¹⁰ ) ₂ , -CON (R ¹¹⁰ ) OR ¹¹⁰ , -OCON (R ¹¹⁰ ) ₂ , -NR ¹¹⁰ COR ¹¹⁰ , -NR ¹¹⁰ CON (R ¹¹⁰ ) ₂ , -NR ¹¹⁰ COOR ¹¹⁰ or -N ( R ¹¹⁰ ) Substituted by 1 to 4 groups of ₂ ;
RN is -L ³¹ - ^{R 60} , where L ³¹ is a bond, -X ³ (CH _z ) _n -X ³ -,-(CH ₂ ) _m -X3- (CH ₂ ) _n -or-. (CH ₂ ) _{1 + w,} -Y ^3- (CH ₂ ) _w- , and in these equations, each w is independently 0-5, and each X ³ is independently combined. , -C (R ¹¹⁰ ) _2- , -C (R ¹¹⁰ ) ₂ C (R ¹¹⁰ ) _2- , -C (R ¹¹⁰ ) = C (R ¹¹⁰ )-, -C≡C-, -CO-,- CS-, -CONR ^100- , -C (= N) (R ¹⁰⁰ )-, -C (= N-OR ¹¹⁰ )-, -C [= N-N (R ¹¹⁰ ) ₂ ], -CO _2- , -SO ₂ -or -SO ₂ N (R ¹¹⁰ )-and Y ³ is -O-, -S-, -NR ^70- , -N (R ¹⁰⁰ ) CO-, -N (R ¹¹⁰ ) CO2-, -OCO-, -OC (= O) N (R ¹⁰⁰ )-, -NR ¹⁰⁰ CONR ^100- , -N (R ¹¹⁰ ) SO _2- or -NR ¹⁰⁰ CSNR ^100- , and
Alternatively, L ³¹ is a C ₂ to C ₆ aligyl chain, provided that the aligyl chain is optionally −C (R ¹¹⁰ ) _2- ,.
-C (R ¹¹⁰ ) ₂ C (R ¹¹⁰ ) _2- , -C (R ¹¹⁰ ) = C (R ¹¹⁰ )-, -C (R ¹¹⁰ ) ₂ O-, -C (R ¹¹⁰ ) ₂ NR ^110- , -C≡C-, -O-, -S-, -N (R ¹⁰⁰ ) CO-,
-N (R ¹⁰⁰ ) CO _2- , -CON (R ¹⁰⁰ )-, -CO-, -CO _2- , -O (C = O)-, -O (C = O) N (R ¹¹⁰ )-, Suspended by -SO _2- , -N (R ¹⁰⁰ ) SO _2- or -SO ₂ N (R ¹⁰⁰ )-
^R60 is C1 to C6 alkyl, C1 to _C6 haloalkyl, aryl, _C3 to _{C8 cycloalkyl ,} heteroaryl, heterocyclyl, _-CN ,
-C (= O) R ¹¹⁰ , -C (= O) OR ¹¹⁰ , -C (= O) N (R ¹¹⁰ ) ₂ , -N (R ¹¹⁰ ) ₂ , -SO ₂ R ¹¹⁰ , -S (= O) ) ₂ N (R ¹¹⁰ ) ₂ , -C (= O) N (R ¹¹⁰ ) N (R ¹¹⁰ ) ₂ or -C (= O) N (R ¹¹ ) (OR ¹¹⁰ ), provided that the aryl, said. Heteroaryl, cycloalkyl or heterocyclyl are optionally substituted with one to four ^R60a , in this case.
Each R ^60a is independently -Z, -Y'-Z or -XY-Z;
Each ^RC is independently -L ³⁰ -R ⁷⁰ , provided that
Each L ³⁰ is independently coupled or − (CH ₂ ) _m −V ¹⁰ − (CH ₂ ) _n −, and in the equation,
V ¹⁰ is -C (R ¹¹⁰ ) _2- , -C (R ¹¹⁰ ) ₂ C (R ¹¹⁰ ) ₂ , -C (R ¹¹⁰ ) = C (R ¹¹⁰ )-, -C (R ¹¹⁰ ) ₂ O- , -C (R ¹¹⁰ ) ₂ NR ^110- , -C≡C-, -O-,
-S-, -NR ^10- , -N (R ¹⁰⁰ ) CO-, -N (R ¹⁰⁰ ) CO2-, -OCO-, -CO-, -CS-, -CONR ^100- , -C (= N-) R ¹¹⁰ )-, -C (= N-OR ¹¹⁰ )-,
-C [= N-N (R ¹¹⁰ ) ₂ ], -CO _2- , -OC (= O)-, -OC (= O) N (R ¹⁰⁰ )-, SO _2- , -N (R ¹⁰⁰ ) SO _2- , -SO ₂ N (R ¹⁰⁰ )-, -NR ¹⁰⁰ CONR ^100- , -NR ¹⁰⁰ CSNR ^100- , C ₃ to C ₆ cycloalkyl or C ₃ to C ₆ cyclohaloalkyl.
Alternatively, each L ³⁰ is independently C ₂ to C ₆ aligyl, provided that the aligyl chain is optionally -C (R ¹¹⁰ ) _2- , -C (R ¹¹⁰ ) ₂ C (R ¹¹⁰ ). _2- , -C (R ¹¹⁰ ) C (R ¹¹⁰ )-, -C (R ¹¹⁰ ) ₂ O-, -C (R ¹¹⁰ ) ₂ NR ^110- , -C≡C-, -O-, -S- , -N (R ¹⁰⁰ ) CO-,
-N (R ¹⁰⁰ ) CO _2- , -NR ^110- , -CON (R ¹⁰⁰ )-, -CO-, -CO _2- , -O (C = O)-, -O (C = O) N ( Suspended by R ¹⁰⁰ )-, -SO _2- , -N (R ¹⁰⁰ ) SO _2- or -SO ₂ N (R ¹⁰⁰ )-
Each R ⁷⁰ is independently hydrogen, halogen, nitro, aryl, heteroaryl, heterocyclyl, -Z, -YZ or -X-YZ.
In this case, the aryl, heteroaryl and heterocyclyl are each optionally substituted with one to four R ^70a , provided that each R ^70a is independent and aryloxy, aralkyloxy, aryloxyalkyl, arylC ₀ . ~ C ₆ alkylcarboxy, C (R ¹¹⁰ ) = C (R ¹¹⁰ ) COOH, oxo, -Z, -Y'-Z or -XY-Z, and each R ^70a is optionally 1 Substituted by four to four R ^80s , provided that each R ⁸⁰ is independently a halogen, C ₁ to C ₆ alkyl, C ₁ to C ₆ alkoxy, C ₁ to C ₈ haloalkyl, C ₁ to C ₈ haloalkyl. (OR ¹¹⁰ ), C ₀ to C ₆ alkyl OR ¹¹⁰ , C ₀ to C ₆ alkyl CON (R ¹¹⁰ ) ₂ , C ₀ to C ₆ alkyl COR ¹¹⁰ , C ₀ to C ₆ alkyl COOR ¹¹⁰ or C ₀ to C ₆ Alkoxy SO ₂ R ¹¹⁰ ;
Each R ¹⁰⁰ is independently -R ¹¹⁰ , -C (= O) R ¹¹⁰ , -CO ₂ R ¹¹⁰ or -SO ₂ R ¹¹⁰ ;
Each R ¹¹⁰ is independently -hydrogen, -C ₁ to C ₆ alkyl, C ₂ to C ₆ alkenyl, -C ₁ to C ₆ alkynyl, -C ₁ to C ₆ haloalkyl or -N (R ¹² ) ₂ . However, any of R ¹¹⁰ is optionally replaced by one to four groups of R ¹²⁰ :
Each R ¹²⁰ is independently halogen, cyano, nitro, oxo, -B (OR ¹³⁰ ), C ₀ to C ₆ alkyl N (R ¹³ ) ₂ , C ₁ to C ₆ haloalkyl, C ₁ to C ₆ alkyl. , C ₁ to C ₆ Alkoxy, (C ₀ to C ₆ Alkoxy) C = O (OR ¹³⁰ ), C ₀ to C ₆ Alkoxy OR ¹³⁰ , C ₀ to C ₆ Alkoxy COR ¹³⁰ ,
C ₀ to C ₆ alkyl SO ₂ R ¹³⁰ , C ₀ to C ₆ alkyl CON (R ¹³ ) ₂ , C ₀ to C ₆ alkyl CONR ¹³⁰ OR ¹³⁰ , C ₀ to C ₆ alkyl SO ₂ N (R ¹³⁰ ) ₂ , C ₀ to C ₆ alkyl SR ¹³⁰ , C ₀ to C ₆ haloalkyl OR ¹³⁰ , C ₀ to C ₆ alkyl CN, -C ₀ to C ₆ Archi N (R ¹³ ) ₂ , -NR ¹³ SO ₂ R ¹³ or -OC _0-6 alkyl COOR ¹³⁰ ;
Each R ¹³⁰ is independently hydrogen, C ₁ to C ₆ alkyl, C ₂ to C ₆ alkenyl or C ₂ to C ₆ alkynyl;
Each R ¹⁴⁰ is independently C ₁ to C ₆ alkyl, C ₁ to C ₆ alkoxy, halogen, C ₁ to C ₆ haloalkyl, C ₀ to C ₆ alkyl CON (R ¹¹⁰ ) _o , C ₀ to C ₆ Alkoxy CONR ¹¹⁰ R ¹⁰ , C ₀ to C ₆ alkyl OR ¹¹⁰ or C ₀ to C ₆ alkyl COOR ¹¹⁰ ; and each R ¹⁵⁰ is independently hydrogen, halogen, OR ¹³⁰ , (C ₁ to C ₆ ). Alkoxy or (C ₁ to C ₆ ) haloalkyl, provided that
In some cases, each alkyl is independently halogen, cyano, nitro, azide, OR ¹³⁰ , C (O) R ¹³⁰ , C (O) OR ¹³ C (O) N (R ¹³⁰ ) ₂ , N (R). ¹³⁰ ) ₂ , N (R ¹³⁰ ) C (O) R ¹³⁰ , N (R ¹³⁰ ) S (O) ₂ R ¹³⁰ , -OC (O) OR ¹³⁰ , OC (O) N (R ¹³⁰ ) ₂ , N ( R ¹³⁰ ) C (O) OR ¹³⁰ , N (R ¹³⁰ ) C (O) N (R ¹³⁰ ), SR ¹³⁰ , S (O) R ¹³⁰ , S (O) ₂ R'or S (O) ₂ N ( R ¹³⁰ ) Substituted by at least one group that is ₂ , or two R ^150s (bonded to the same or different atoms) can be combined to form a C3-C6 cycloalkyl;
Each X is independently -O-, -S- or -N (R ¹⁰⁰ )-;
Each Y is independently- [C (R ¹⁵⁰ ) ₂ ] _p- or -C ₂ to C ₆ alkenyl, where p is 1, 2, 3, 4, 5 or 6;
Each Y'is independently- [C (R ¹⁵⁰ ) ₂ ] _p- , -C ₂ to C ₆ alkenyl C ₃ to C ₈ cycloalkyl or heterocyclyl, provided that the cycloalkyl or heterocyclyl is optionally. Substituted by one to three Z groups:
Each Z is independently -H, halogen, -OR ¹¹⁰ , -SR ¹¹⁰ , -C (= O) R ¹¹⁰ , -C (= O) OR ¹¹⁰ , -C (= O) N (R ¹¹⁰ ). ₂ ,
-N (R ¹⁰⁰ ) ₂ , -N ₃ , -NO ₂ , -C (= N-OH) R ¹¹⁰ , -C (= S) N (R ¹¹⁰ ) ₂ , -CN, -S (= O) R ¹¹⁰ , -S (= O) N (R ¹¹⁰ ) ₂ , -S (= O) OR ¹¹⁰ ,
-S (= O) ₂ R ¹¹⁰ , S (= O) ₂ N (R ¹¹⁰ ) ₂ , -NR ¹¹⁰ COR ¹¹⁰ , -N (R ¹¹⁰ ) C (= O) N (R ¹¹⁰ ) ₂ , -N ( R ¹¹⁰ ) COOR ¹¹⁰ , -N (R ¹¹⁰ ) S (= O) ₂ R ¹¹⁰ , -C (= O) N (R ¹¹⁰ ) N (R ¹¹⁰ ) ₂ , -C (= O) N (R ¹¹⁰ ) (OR ¹¹⁰ ), -OC (= O) -R ¹¹⁰ , -OC (= O) -OR ¹¹⁰ or -OC (= O) -N (R ¹¹⁰ ) ₂ ; and each m and n are independent. 0, 1, 2, 3, 4, 5 or 6.

他の実施形態において、式ＶＩの化合物は下記の式ＶＩＩの構造を有する：

さらに他の実施形態において、式ＶＩの化合物は下記の式ＶＩＩＩの構造を有する：

なおもさらなる実施形態において、式ＶＩの化合物は下記の式ＩＸの構造を有する：

したがって、本発明の方法において有用であり得る式ＩＶの化合物には、下記に示される構造を有する化合物およびそれらのその医薬的に許容される塩が含まれるが、これらに限定されない：

また、本発明の方法において有用であり得る式ＩＶの化合物は、下記の化合物を含むリストから選択される：
３３、２－（１－（３クロロ－３’－フルオロ－４’－（ヒドロキシメチル）－５’－（メチルスルホニル）ビフェニル－４－イル）－２－（２－（２，６ジクロロフェニル）プロパン－２－イル）－１Ｈ－イミダゾール－４－イル）プロパン－２－オール；３４、２－（２－（２（２－クロロ－３－フルオロフェニル）プロパン－２－イル）－１－（３’－フルオロ－４’－（ヒドロキシメチル）－５’（メチルスルホニル）ビフェニル－４－イル）－１Ｈ－イミダゾール－４－イル）プロパン－２－オール；３５、２－（２－（２（２，６－ジクロロフェニル）プロパン－２－イル）－１－（３’－フルオロ－４’－（ヒドロキシメチル）－５’（メチルスルホニル）ビフェニル－４－イル）－１Ｈ－イミダゾール－４－イル）プロパン－２－オール；３６、２－（２－（２（２，６－ジクロロフェニル）プロパン－２－イル）－１－（３，３’－ジフルオロ－４’－（ヒドロキシメチル）－５’（メチルスルホニル）ビフェニル－４－イル）－１Ｈ－イミダゾール－４－イル）プロパン－２－オール；および、３７、２－（２－［１（２，６－ジクロロフェニル
）エチル］－１－［３，３’－ジフルオロ－４’－（ヒドロキシメチル）－５’（メチルスルホニル）ビフェニル－４－イル］－１Ｈ－イミダゾール－４－イル）プロパン－２－オール。化合物１２はまた、ＷＯ２０１０ ０１３８５９８のＥｘ．９として知られている。化合物３８はまた、ＷＯ２００７ ００２５６３のＥｘ．１９として知られている。化合物３９はまた、ＷＯ２０１２ ０１３５０８２として知られている。 In some embodiments, the compound of formula IV has the structure of formula V or formula VI below:

In other embodiments, the compound of formula VI has the structure of formula VII below:

In yet another embodiment, the compound of formula VI has the structure of formula VIII below:

Still in further embodiments, the compound of formula VI has the structure of formula IX below:

Accordingly, compounds of formula IV that may be useful in the methods of the invention include, but are not limited to, compounds having the structures shown below and their pharmaceutically acceptable salts:

Also, compounds of formula IV that may be useful in the methods of the invention are selected from the list containing the following compounds:
33,2- (1- (3chloro-3'-fluoro-4'-(hydroxymethyl) -5'-(methylsulfonyl) biphenyl-4-yl) -2- (2- (2,6 dichlorophenyl) propane) -2-yl) -1H-imidazol-4-yl) Propan-2-ol; 34,2-(2-(2 (2-chloro-3-fluorophenyl) propan-2-yl) -1- (3) '-Fluoro-4'-(hydroxymethyl) -5'(methylsulfonyl) biphenyl-4-yl) -1H-imidazole-4-yl) propan-2-ol; 35,2- (2- (2 (2) , 6-Dichlorophenyl) Propan-2-yl) -1- (3'-Fluoro-4'-(hydroxymethyl) -5'(methylsulfonyl) biphenyl-4-yl) -1H-imidazole-4-yl) propane -2-ol; 36,2- (2- (2 (2,6-dichlorophenyl) propan-2-yl) -1- (3,3'-difluoro-4'-(hydroxymethyl) -5'(methyl) Methyl) biphenyl-4-yl) -1H-imidazol-4-yl) propan-2-ol; and 37,2-(2- [1 (2,6-dichlorophenyl) ethyl] -1- [3,3 '-Difluoro-4'-(hydroxymethyl) -5'(methylsulfonyl) biphenyl-4-yl] -1H-imidazole-4-yl) propan-2-ol. Compound 12 also includes Ex. Known as 9. Compound 38 is also described in WO2007 002563 Ex. Known as 19. Compound 39 is also known as WO2012 0135802.

Compounds of formula IV can be synthesized as described in PCT Publication No. US2010 / 0069367 and WO2010 / 138598, which are incorporated herein by reference.

さらなる実施形態において、転移の処置および／または防止のために使用することができる化合物が、下記からなるリストにおけるＰＣＴ公開公報において見出され得る：ＷＯ２００６／０９４０３４、ＷＯ２００８／０４９０４７、ＷＯ２００９／０２０６８３、ＷＯ２００９／０８６１３８、ＷＯ２００９／０８６１２３、ＷＯ２００９／０８６１３０、ＷＯ２００９／０８６１２９、ＷＯ２００７／００２５５９、ＷＯ２００７／００２５６３、ＷＯ２００７／０８１３３５、ＷＯ２００６／０１７０５５、ＷＯ２００６／１０２０６７、ＷＯ２００９／０２４５５０、ＵＳ２００６／００７４１１５、ＵＳ２００６／０１３５６０１、ＷＯ２００９／０２１８６８、ＷＯ２００９／０４０２８９、ＷＯ２００７／０４７９９１、ＷＯ２００７／０５０４２５、ＷＯ２００６／０７３３６３、ＷＯ２００６／０７３３６４、ＷＯ２００６／０７３３６５、ＷＯ２００６／０７３３６６、ＷＯ２００６／０７３３６７、ＵＳ２００９／００３００８２、ＷＯ２００８／０６５７５４、ＪＰ２００８／１７９５６２、ＷＯ２００７／０９２０６５、ＵＳ２０１０／００６９３６７、ＵＳ７９９８９９５、ＵＳ７２４７７４８、ＷＯ２０１０／１３８５９８、ＵＳ７３６５０８５、ＵＳ７５７７６２１５、ＵＳ６３１３６５０３、ＵＳ２００４／００７２８６８、ＵＳ２００５／０１０７４４４、ＵＳ２００５／０１１３５８０、ＵＳ２００５／０１３１０１４、ＵＳ２００５／０２８２９０８、ＵＳ２００９／０２８６７８０（これらは参照によって本明細書中に組み込まれる）。 The LXR agonist that can be used for the treatment and / or prevention of metastasis can be compound 24 or a pharmaceutically acceptable salt thereof:

In a further embodiment, compounds that can be used for the treatment and / or prevention of metastasis can be found in the PCT Publications in the list consisting of: WO2006 / 094034, WO2008 / 049047, WO2009 / 020683, WO2009. / 086138, WO2009 / 086123, WO2009 / 086130, WO2009 / 086129, WO2007 / 002559, WO2007 / 00263, WO2007 / 081335, WO2006 / 017055, WO2006 / 10267, WO2009 / 024550, US2006 / 0074115, US2006 / 0135601, WO , WO2009 / 040289, WO2007 / 047991, WO2007 / 050425, WO2006 / 073363, WO2006 / 073364, WO2006 / 073365, WO2006 / 073366, WO2006 / 073367, US2009 / 0030082, WO2008 / 065754, JP2008 / 17956 / 0069367, US7799895, US722748, WO2010 / 138598, US7365085, US75776215, US63136503, US2004 / 0072868, US2005 / 0107444, US2005 / 0113580, US2005 / 0131014, US2005 / 0282808, US2009 / 0286780. Will be incorporated).

LXRα and LXRβ were first discovered by a large number of groups at about the same time (Apfeld et al., 1994; Willy et al., 1995; Song et al., 1994; Shinar et al., 1994; Teboul et al., 1995), Glucose,. It belongs to a family of nuclear hormone receptors that are endogenously activated by cholesterol and its oxidative derivatives to mediate the transcription of genes involved in the metabolism of cholesterol and fatty acids (Janowski et al., 1996; Calkin and Totonoz, 2012). Given the intricate link between lipid metabolism and cancer cell growth (Cairns et al., 2011), the ubiquitous expression of LXRβ in melanoma is not considered to be a coincidence, thereby causing melanoma cells. This is because lipid and lipoprotein particles can be synthesized to sustain their growth. However, at the same time, such stable underlying expression levels make LXRβ an ideal therapeutic target, as illustrated by the widespread responsiveness of melanoma cells to LXRβ activation therapy.

Various compounds have been shown to have selectivity for LXRβ or LXRα. This selectivity may allow for increased activity and / or decreased off-target effects. Examples of compounds having selectivity for LXRβ or LXRα are shown in Table 1.

本明細書中で使用される場合、ＬＸＲαおよびＬＸＲβにおけるＬＸＲアゴニストの活性に対する参照は、Ｓｐｅｎｃｅｒ他、Ｊｏｕｒｎａｌ ｏｆ Ｍｅｄｉｃｉｎａｌ Ｃｈｅｍｉｓｔｒｙ、２００１、４４、８８６～８９７（これは参照によって本明細書中に組み込まれる）に記載されるリガンドセンシングアッセイ（ＬｉＳＡ）を使用して測定されるような活性を示す。いくつかの実施形態において、ＬＸＲアゴニストは、リガンドセンシングアッセイにおいて１μＭ未満のＥＣ５０（例えば、０．５ｎｍ～５００ｎＭ、１０ｎＭ～１００ｎＭ）を有する。例えば、本発明の方法は、ＬＸＲαに対する当該アゴニストの活性よりも少なくとも３倍大きいＬＸＲβに対する活性を有するＬＸＲβアゴニスト、または、ＬＸＲαに対する当該アゴニストの活性よりも少なくとも１０倍大きいＬＸＲβに対する活性を有するＬＸＲβアゴニスト、または、ＬＸＲαに対する当該アゴニストの活性よりも少なくとも１００倍大きいＬＸＲβに対する活性を有するＬＸＲβアゴニスト、または、ＬＸＲαに対する当該アゴニストの活性の少なくとも３倍以内であるＬＸＲβに対する活性を有するＬＸＲβアゴニストを使用して行うことができる。ＬｉＳＡアッセイアッセイにおける「より大きい活性」という用語は、より低いＥＣ_５０を示す。例えば、ＧＷ３９６５［２］は、ＬＸＲα（ＥＣ_５０＝１９０）と比較して、ＬＸＲβ（ＥＣ_５０＝３０）に対するおよそ６倍より大きい活性を有する。

As used herein, references to the activity of LXR agonists in LXRα and LXRβ include Spencer et al., Journal of Medicinal Chemistry, 2001, 44, 886-897 (which are incorporated herein by reference). Shows activity as measured using the ligand sensing assay (LiSA) described in. In some embodiments, the LXR agonist has an EC50 of less than 1 μM (eg, 0.5 nm to 500 nM, 10 nM to 100 nM) in the ligand sensing assay. For example, the method of the present invention comprises an LXRβ agonist having an activity on LXRβ that is at least 3 times greater than the activity of the agonist on LXRα, or an LXRβ agonist having an activity on LXRβ that is at least 10 times greater than the activity of the agonist on LXRα. Alternatively, use an LXRβ agonist having an activity against LXRβ that is at least 100 times greater than the activity of the agonist against LXRα, or an LXRβ agonist having an activity against LXRβ that is at least 3 times the activity of the agonist against LXRα. Can be done. LiSA Assay The term "greater activity" in an assay indicates a lower _EC50 . For example, GW3965 [2] has approximately 6-fold greater activity against LXRβ (EC ₅₀ = 30) as compared to LXRα (EC ₅₀ = 190).

As used herein, the term "increasing the level of ApoE expression in vitro" means that a particular LXR agonist raises the level of ApoE expression to a concentration of less than 5 μM (eg, a concentration of 100 nM to 2 μM, 1 μM or less). (Concentration) can be increased 2.5-fold in the qPCR assay of Example 21. LXR agonists exhibiting this in vitro effect can be very effective for use in the methods of the invention.

The term "alkyl" as used in this application relates to saturated branched or non-branched aliphatic monovalent substituents. Alkyl substituents are 1 to 100 carbon atoms (for example, 1 to 22 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, 1 to 3 carbon atoms). ). Thus, examples of alkyl substituents include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl.

The term "alkoxy" represents a chemical substituent of formula-OR, where R is an optionally substituted C1-C6 alkyl group unless otherwise specified. In some embodiments, the alkyl group can be substituted, for example, the alkoxy group comprises one, two, three, four, substituents as defined herein. You can have 5 or 6.

The term "alkoxyalkyl" refers to a heteroalkyl group described as an alkyl group substituted with an alkoxy group, which is as defined herein. An exemplary unsubstituted alkoxyalkyl group contains between 2 and 12 carbons. In some embodiments, alkyl and alkoxy can each be further substituted with one, two, three or four substituents as defined herein for each group.

As used herein, the term "cycloalkyl" refers to monocyclic, bicyclic or tricyclic substituents, provided that they may be saturated or partially saturated. It may be saturated, i.e., with one or more double bonds. The monocyclic substituent is exemplified by a saturated cyclic hydrocarbon group containing 3 to 8 carbon atoms. Examples of monocyclic cycloalkyl substituents include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl and cyclooctyl. Bicyclic fused cycloalkyl substituents are exemplified by cycloalkyl rings that condense to another cycloalkyl ring. Examples of bicyclic cycloalkyl substituents include, but are not limited to, decalin and 1,2,3,7,8,8a-hexahydro-naphthalene. A tricyclic cycloalkyl substituent is exemplified by a cycloalkyl bicyclic fused ring that condenses with a further cycloalkyl substituent.

The term "alkylene" as used in this application relates to saturated branched or non-branched aliphatic divalent substituents (eg, an alkylene substituent is one to six carbon atoms, one. It has ~ 3 carbon atoms). Thus, examples of alkylene substituents include methylene, ethylene, trimethylene, propylene, tetramethylene, isopropylene, pentamethylene and hexamethylene.

The term "alkenylene or alkenyl", as used in this application, is an unsaturated branched or non-branched aliphatic divalent substituent having a double bond between two adjacent carbon atoms ( For example, an alkenylene substituent has 2 to 6 carbon atoms (2 to 4 carbon atoms). Therefore, examples of alkenylene substituents include vinylene, 1-propenylene, 2-propenylene, methylvinylene, 1-butenylene, 2-butenylene, 3-butenylene, 2-methyl-1-propenylene, 2-methyl-2-propenylene. , 2-Pentenylene, 2-Hexenylene, but not limited to these.

The term "alkynylene or alkynyl", as used in this application, is an unsaturated branched or non-branched aliphatic divalent substituent having a triple bond between two adjacent carbon atoms (eg,). , The alkynylene substituent has 2 to 6 carbon atoms and 2 to 4 carbon atoms). Examples of alkynylene substituents include, but are not limited to, ethynylene, 1-propinylene, 1-butynylene, 2-butynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene and 2-hexynylene.

The term "alkazienylene", as used in this application, is an unsaturated branched or non-branched aliphatic divalent substituent having two double bonds between two adjacent carbon atoms (). For example, an alkadienylene substituent has 4 to 10 carbon atoms). Therefore, examples of alkazienylene substituents include 2,4-pentadienylene, 2,4-hexadienylene, 4-methyl-2,4-pentadienylene, 2,4-heptadienylene, 2,6-heptadienylene, 3-methyl-2, 4-Hexadienylene, 2,6-octadienylene, 3-methyl-2,6-heptadienylene, 2-methyl-2,4-heptadienylene, 2,8-nonadienylene, 3-methyl-2,6-octadienylene, 2,6- Includes, but is not limited to, the substituents of decadienylene, 2,9-decadienylene and 3,7-dimethyl-2,6-octadienylene.

As used herein, the term "heteroaliphatic substituent or heteroalkyl" means that one or more carbon atoms are heteroatoms, eg, oxygen atom, sulfur atom, nitrogen atom, phosphorus atom or silicon. Indicates a monovalent or divalent substituent substituted by an atom, provided that the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. In some cases. The heteroatoms O, N and S may be placed at any internal position of the heteroaliphatic substituent. For example, -CH ₂ -CH ₂ -O-CH ₃ , -CH ₂ -CH ₂ -NH-CH ₃ , -CH ₂ -CH ₂ -N (CH ₃ ) -CH ₃ , -CH ₂ -S- CH ₂ -CH ₃ , -S (O) -CH ₃ , -CH ₂ -CH ₂ -S (O) ₂ -CH ₃ , -CH = CH-O-CH ₃ , -CH ₂ -CH = N-OCH ₃ and -CH = CH-N (CH ₃ ) -CH ₃ are included. Heteroaliphatic substituents may be linear or branched and saturated or unsaturated.

In one embodiment, the heteroaliphatic substituent has 1 to 100 (eg, 1 to 42 carbon atoms). In yet another embodiment, the heteroaliphatic substituent is a polyethylene glycol residue.

As used herein, the term "aromatic substituent or aryl" is 10 in which at least one ring is aromatic and may be unsubstituted or substituted. It is intended to mean any stable monocyclic, bicyclic or polycyclic carbocycle with atoms up to in each ring. Examples of such aromatic substituents include phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. If the aromatic substituent is bicyclic and one ring is non-aromatic, it is understood that the bond is via an aromatic ring.

The term "alkylaryl substituent or arylalkyl" is described above where one or more bonds to the hydrogen contained are replaced by a bond to any of the aryl substituents as described above. Such alkyl substituents are shown. It is understood that arylalkyl substituents lead to carbonyl groups in the compounds of the invention via bonds from alkyl substituents. Examples of arylalkyl substituents are benzyl (phenylmethyl), p-trifluoromethylbenzyl (4-trifluoromethylphenylmethyl), 1-phenylethyl, 2-phenylethyl, 3-phenylpropyl and 2-phenylpropyl. Etc., but not limited to these.

The term "heteroaromatic substituent or heteroaryl", as used herein, is one in which at least one ring is aromatic and is selected from the group consisting of O, N and S. Represents a stable monocyclic, bicyclic or polycyclic ring containing up to 10 atoms, each containing 4 heteroatoms. Bicyclic heteroaromatic substituents include
a) Condensate into a 6-membered aromatic (unsaturated) heterocyclic ring with one nitrogen atom,
b) Condensate into a 5- or 6-membered aromatic (unsaturated) heterocyclic ring with two nitrogen atoms, c) Whether one nitrogen atom is one oxygen atom or one sulfur atom A 5-membered aromatic (unsaturated) heterocyclic ring that is fused to a 5-membered aromatic (unsaturated) heterocyclic ring with a sword, or d) a 5-membered aromatic (unsaturated) heteroatom having one heteroatom selected from O, N and S. Includes a phenyl ring, a pyridine ring, a pyrimidine ring or a pyrididine ring fused to a cyclic ring.

Heteroaryl groups within the scope of this definition include, but are not limited to, the following groups: benzoimidazolyl, benzofuranyl, benzofrazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazoline. , Carbazolyl, Carborinyl, Synnolinyl, Furanyl, Indolinyl, Indrill, Indolazinyl, Indazolyl, Isobenzofuranyl, Isoindrill, Isoquinolyl, Isothiazolyl, Isoxazolyl, Naftpyridinyl, Oxazodiazolyl, Oxazoline, Oxazoline, Isooxazolyl, Oxazoline, Pyrazoline, Pyrazoline , Pyridopyridinyl, pyridadinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxazoline, tetrazolyl, tetrazolopyridyl, thiathiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, aziridinyl, 1,4-dioxanyl, hexahydroazepinyl, dihydrobenzoidazolyl Dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazoline, dihydrofuranyl, dihydroimidazolyl, dihydroindrill, dihydroisoxazoline, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazoline, dihydropi Radinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazoline, dihydrothiadiozolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidini Lu, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indrill, benzotriazolyl, benzothiazolyl, benzoxazoline, isooxazoline, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, Quinoline, isoquinolinyl, oxazoline, isooxazoline, indolyl, pyrazinyl, pyridadinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. If the heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain a heteroatom, the bonds will be via the aromatic ring or heteroatom, respectively. It is understood that it is through the contained ring. If the heteroaryl contains a nitrogen atom, it is understood that the corresponding N-oxide is also included by the present invention.

Aryl substituents, heteroaliphatic substituents, aromatic substituents and heteroaromatic substituents may optionally be by one or more of the following substituents, including but not limited to the various groups below. Can be substituted one or more times in the same or different manners: aliphatic substituents, heteroaliphatic substituents, aromatic substituents and heteroaromatic substituents, aryls, heteroaryls; alkylaryls; heteroalkylaryls. Alkyl Heteroaryl; Heteroalkyl Heteroaryl; Alkique; Aryloxy; Heteroalkoxy; Heteroaryloxy; Alkylthio; Arylthio; Heteroalkylthio; Heteroarylthio; F; Cl; Br; I; -OH; -NO ₂ ; -CN -CF ₃ ; -CH ₂ CF ₃ ; -CHCl ₂ ; -CH ₂ OH; -CH ₂ CH ₂ OH; -CH ₂ NH ₂ ; -CH ₂ SO ₂ CH ₃ ; -C (O) R _x ;- CO ₂ (R _x ); -CON (R _x ) ₂ ; -OC (O) R _x ; -OCO ₂ R _x ; -OCON (R _x ) ₂ ; -N ( _RX ) ₂ ; -S (O) R _x ; -S (O) ₂ R _x ; -NR _x (CO) R _x , however, in these equations, the presence of each of R _x is independent of the aliphatic, alicyclic, and heterolipids. , Heterocyclic, Aromatic, Heteroaromatic, Aryl, Heteroaryl, Alkylaryl, Alkyl Heteroaryl, Heteroalkylaryl or Heteroalkyl Heteroaryl, but is not limited to these, as described above and herein. Any of the aliphatic substituents, alicyclic substituents, heteroaliphatic substituents, heterocyclic substituents, alkylaryl substituents or alkylheteroaryl substituents described in the book may be substituted or unsubstituted. It may be branched or non-branched, saturated or unsaturated, and may be an aromatic substituent, a heteroaromatic substituent, an aryl substituent, or a heteroaryl substituted as described above and herein. Any of the groups, (alkyl) aryl substituents or (alkyl) heteroaryl substituents may be substituted or unsubstituted. In addition, any two adjacent substituents together form a substituted or unsubstituted 4-membered, 5-membered, 6-membered or 7-membered alicyclic or heterocyclic substituent. It will be understood that it may be represented. Further examples of commonly applicable substituents are exemplified by the specific embodiments shown below.

The terms "halo" and the term "halogen" refer to halogen atoms selected from the group consisting of F, Cl, Br and I.

The term "halogenated alkyl substituent, haloalkyl" refers to an alkyl substituent as defined above, which is substituted with at least one halogen atom. In one embodiment, the alkyl halide substituent is perhalogenated. In another embodiment, the perfluoroalkyl represents an alkyl halide substituent which is a monovalent perfluoroylated substituent of the formula C _n F _{2n + 1} . For example, an alkyl halide substituent may have 1 to 6 carbon atoms (eg, 1 to 3 carbon atoms). Thus, examples of alkyl groups include trifluoromethyl, 2,2,2-trifluoroethyl, n-perfluoropropyl, n-perfluorobutyl and n-perfluoropentyl.

The term "amino", as used herein, stands for -N (RN1) ₂ , but in the formula, each ^RN1 is independently H, OH, NO ₂ , ^N (R). ^N2 ) ₂ , SO ₂ OR ^N2 , SO ₂ RN2, SOR ^N2 , ^N -protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkalil, cycloalkyl, alkcycloalkyl, heterocyclyl (eg, hetero). Aryl), alcoholicryl (eg, alkylheteroaryl), or two ^RN1s together to form a heterocyclyl or ^N -protecting group, each of which has an RN2. Independently, it is H, alkyl or aryl. In a preferred embodiment, the amino is -NH ₂ or -NHR ^N1 , where ^RN1 is independently OH, NO ₂ , NH ₂ , NR ^N2 ₂ , SO ₂ OR ^N2 , SO ₂ ^RN 2 in the formula. , SOR ^N2 , alkyl or aryl, and each ^RN2 can be H, alkyl or aryl. The term "aminoalkyl", as used herein, is an alkyl group (which is defined herein) substituted with an amino group (which is as defined herein). Represents a heteroalkyl group described as (as defined herein). Alkyl and amino can each be further substituted with one, two, three or four substituents as described herein for each group. For example, the alkyl moiety may contain an oxo (= O) substituent.

As used herein, the term "aryloxy" refers to an aromatic or heteroaromatic system that is linked to another residue via an oxygen atom. A typical example of O-aryl is phenoxy. Similarly, an "arylalkyl" is via a carbon chain (saturated or unsaturated) (including its heteromorphic form), typically a C1-C8 carbon chain and a C1-C6 carbon chain when saturated. Or, more specifically, via the carbon chains of C1 to C4 or the carbon chains of C1 to C3 (including their heteromorphic forms), or, when unsaturated, the carbon chains of C2 to C8, C2 to C6. Indicates an aromatic or heteroaromatic system linked to another residue via a carbon chain of C2 to C4 or a carbon chain of C2 to C3 (including their heteromorphic forms). More certainly, therefore, arylalkyl is defined above as being linked to an alkyl component, a heteroalkyl component, an alkenyl component, a heteroalkenyl component, an alkynyl component or a heteroalkynyl component as also defined above. Aryl groups or heteroaryl groups are included. Typical arylalkyls include aryl (C6-C12) alkyl (C1-C8), aryl (C6-C12) alkenyl (C2-C8) or aryl (C6-C12) alkynyl (C2-C8), in addition. Those heteromorphic forms will be mentioned. A typical example is phenylmethyl, which is commonly referred to as benzyl.

Typical substituents as required in aromatic or heteroaromatic groups include halo, CN, NO ₂ , CF ₃ , OCF ₃ , COOR', _CONR'2 , OR', SR', SOR'. , SO ₂ R', NR' ₂ , NR'(CO) R', NR'C (O) OR', NR'C (O) NR' ₂ , NR'SO ₂ NR' ₂ or NR'SO ₂ R 'Is included independently, where each R'is independently H, or alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and aryl (all of these). It is a optionally substituted group selected from (as defined above); or the substituents are alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, O-. A optionally substituted group selected from aryl, O-heteroaryl and arylalkyl.

Optional substituents on non-aromatic groups (eg, alkyl, alkenyl and alkynyl groups) are typically aromatic or hetero, except as otherwise described herein. Selected from the same list of preferred substituents for aromatic groups. Non-aromatic groups also include = O and = NOR'(where R'in the formula is H, or alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and aryl (all of these). It may contain substituents selected from (as defined above), which are optionally substituted groups).

In general, substituents (eg, alkyl, alkenyl, alkynyl or aryl (including all heteroforms defined above)) may themselves be optionally substituted with additional substituents. The properties of these substituents are similar to those listed for substituents in the basic structure described above. Thus, if one embodiment of a substituent is an alkyl, the alkyl is optionally the rest listed as a substituent in which the substitution has chemical significance and the substitution does not compromise the size limit of the alkyl itself. May be substituted with substituents; for example, alkyl substituted with alkyl or with alkenyl is not considered and is not included, as it is believed that for these embodiments it would simply extend the upper limit of the carbon atom. However, alkyl substituted with aryls, aminos, halos, etc. will be included. For example, when a group is substituted, the group may be substituted with one, two, three, four, five or six substituents. Substituents as required include, but are not limited to, the following groups: C1-C6 alkyl or heteroaryl, C2-C6 alkenyl or heteroalkenyl, C2-C6 alkynyl or heteroalkynyl, halogen; aryl, Heteroaryl, azide (-N ₃ ), nitro (-NO ₂ ), cyano (-CN), acyloxy (-OC (= O) R'), acyl (-C (= O) R'), alkoxy (- OR'), amide (-NR'C (= O) R'' or -C (= O) NRR'), amino (-NRR'), carboxylic acid (-CO ₂ H), carboxylic acid ester (-CO) ₂ R'), carbamoyl (-OC (= O) NR'R'' or -NRC (= O) OR'), hydroxy (-OH), isocyano (-NC), sulfonate (-S (= O) ₂ ) OR), sulfonamide (-S (= O) ₂ NRR'or -NRS (= O) ₂ R') or sulfonyl (-S (= O) ₂ R), but in these equations the respective R or R'is independently selected from H, C1-C6 alkyl or heteroaryl, C2-C6 alkenyl or heteroalkenyl, 2C-6C alkynyl or heteroalkynyl, aryl or heteroaryl. The substituted group may have, for example, one, two, three, four, five, six, seven, eight or nine substituents.

The term "heterocyclyl, heterocyclic or Het", as used herein, is independently selected from the group consisting of nitrogen, oxygen and sulfur, unless otherwise specified 1
Represents a cyclic heteroalkyl or heteroalkenyl containing, 2, 2, 3 or 4 heteroatoms, eg, a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring or a 7-membered ring. The 5-membered ring has 0 to 2 double bonds, and the 6- and 7-membered rings have 0 to 3 double bonds. The term "heterocyclyl" also refers to a heterocyclic compound having a crosslinked polycyclic structure in which one or more carbons and / or heteroatoms crosslink two non-adjacent members of a monocyclic ring, eg, for example. Represents a quinuclidinyl group. The term "heterocyclyl" refers to any one of the above heterocyclic rings being one, two or three carbocyclic rings (eg, aryl ring, cyclohexane ring, cyclohexene ring, cyclopentane ring, cyclopentane ring) or another. Includes bicyclic, tricyclic and tetracyclic groups that condense on monocyclic heterocyclic rings such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl and benzothienyl.

Some of the compounds of the invention may contain one or more stereogen centers and may therefore be present in various isomeric forms, such as stereoisomers and / or diastereomers. Accordingly, the compounds of the invention and their pharmaceutical compositions may be in the form of individual enantiomers, diastereomers or geometric isomers, or may be in the form of mixtures of stereoisomers. In certain embodiments, the compounds of the invention are enantiopure compounds. In certain other embodiments, a mixture of stereoisomers or diastereomers is provided. Moreover, when the compounds of the invention are present in tautomeric form, each tautomer is included herein.

Further, as described herein, if the particular compound has one or more double bonds that may be present as either Z isomers or E isomers, unless otherwise indicated. There is. The invention further comprises the compound as an individual isomer substantially free of other isomers and, in the alternative, as a mixture of various isomers, eg, a racemic mixture of stereoisomers. In addition to the above-mentioned compounds themselves, the invention also includes pharmaceutically acceptable derivatives of these compounds, and one or more compounds of the invention, and one or more pharmaceutically acceptable excipients. Includes compositions containing a form or additive.

Treatment Methods As disclosed herein, miR-1908, miR-199a-3p, miR-199a-5p and CTGF are endogenous metastatic promoters of metastatic infiltration, endothelial recruitment and colony formation in melanoma. On the other hand, DNAJA4, ApoE, LRP1, LRP8, LXR and miR7 function as metastasis inhibitors or metastasis inhibitors of the same process. In addition, it was found that these miRNAs convergently target ApoE and the heat shock factor DNAJA4. ApoE secreted by cancer suppresses infiltration and endothelial recruitment by activating the LRP1 receptor on melanoma cells and the LRP8 receptor on endothelial cells, respectively. As a result, DNAJA4 induces ApoE expression. These miRNAs strongly predict the outcome of metastasis in humans. Pretreatment with locked nucleic acids (LNAs) targeting miR-199a-3p, miR-199a-5p and miR-1908 inhibits metastasis to multiple organs, whereas therapeutic delivery of these LNAs Metastasis of human melanoma cells in a mouse model is significantly suppressed.

Accordingly, the present invention provides a method for treating melanoma through increasing the expression or activity level of one of the metastatic inhibitors in a subject. This increase can be achieved, among other things, by forced expression of one or more of the metastatic inhibitors DNAJA4, ApoE, LRP1 and LRP8, or by miR-199a-3p, miR-199a-5p and miR-1908. It can be achieved by reducing one or more expression levels or activity levels. In addition, treatment can be accomplished by reducing the level of expression or activity of one or more of the metastasis-promoting factors.

The present invention also provides methods for treating angioplastic disorders or disorders of angioplasty in a subject. The terms "angiogenic disorders", the terms "angiogenic disorders" and the terms "angiogenic disorders" are used interchangeably herein to indicate disorders characterized by pathological angiogenesis. Disorders characterized by pathological angioplasty are disorders in which abnormal or abnormal angioplasty alone or in combination contributes to the cause, initiation or symptoms of the disorder. show. Examples of this disorder include various cancers (eg, angioplasted tumors), eye disorders and inflammatory disorders.

Typical angioplastic tumors that can be treated by the above methods include solid tumors (particularly cancer tumors) that require vascular components to provide oxygen and nutrients. Exemplary solid tumors include lung, breast, bone, ovary, stomach, pancreas, pharynx, esophagus, testis, liver, parotid gland, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, Includes, but is limited to, bladder, prostate, thyroid cancer, squamous epithelial cancer, adenocarcinoma, small cell carcinoma, melanoma, glioma, glioblastoma, neuroblastoma, Kaposi sarcoma and sarcoma. Not done.

Many disorders or conditions other than cancer can also be treated by the methods described above. Examples include arthritis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, age-related yellow spot degeneration, Graves' disease, vascular restenosis (including restenosis after angiogenesis), arteriovenous malformations (AVM). ), Mental tumor, hemangiomas, angiogenic glaucoma, chronic kidney disease, diabetic nephropathy, multiple cystic kidney disease, interstitial lung disease, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), emphysema, Includes autoimmune hepatitis, chronic inflammatory liver disease, liver cirrhosis, cutaneous T-cell lymphoma, alcohol and basal cell cancer.

Other treatment targets include, for example, those described in US Patent Application Publication Nos. 2009004297, 20090175791 and 20070161553, eg, vascular fibroma, atherosclerotic plaque, corneal transplant angiogenesis, blood. Familiar joints, hypertrophic scars, Osler-Weber syndrome, pyogenic granuloma, posterior crystalline fibroproliferative disorder, scleroderma, tracoma, vascular adhesions, synovitis, dermatitis, various other inflammatory diseases and inflammation Includes sexual disorders as well as endometriosis.

Forced Expression of Metastasis Inhibitors Both the above transfer inhibitor polypeptides (eg, DNAJA4, ApoE, LRP1, LRP8 and LXR) and nucleic acids encoding these polypeptides are used to carry out the present invention. can do. Many polypeptide preparations can be used, but highly purified or isolated polypeptides are preferred. The terms "peptide", term "polypeptide" and term "protein" are used interchangeably herein to describe the sequence of amino acid residues in a polymer. Peptides, polypeptides or proteins can be composed of 20 standard naturally occurring amino acids in addition to rare amino acids and synthesized amino acid analogs. The peptide, polypeptide or protein can be any chain of amino acids, regardless of length or post-translational modification (eg, glycosylation or phosphorylation).

"Inventive" polypeptides include fusions or chimeras produced by recombination or synthesis of any of the above-mentioned metastasis-suppressing factors having specific domains or moieties involved in the network. The term also includes polypeptides with added amino-terminal methionine (useful for expression in prokaryotic cells).

Within the scope of the invention are fusion proteins containing one or more of the above sequences and heterologous sequences. "Chimera" or "fusion" indicates that amino acid sequences of different origin are combined in one polypeptide chain by a combined reading frame of their encoding nucleotide sequences. The term explicitly includes the insertion of an internal fusion, i.e., a sequence of different origin within a polypeptide chain, in addition to fusion to one of its ends. A heterologous polypeptide, nucleic acid or gene is derived from a foreign body species or, if derived from the same species, is substantially modified from its original form. The two fused domains or sequences are heterologous to each other if they are not adjacent to each other in a naturally occurring protein or nucleic acid.

An "isolated" or "purified" polypeptide refers to a polypeptide that has been isolated from other naturally associated proteins, lipids and nucleic acids. The polypeptide is at least 10% by dry weight of the purified preparation (ie, any percentage between 10% and 100%, eg, 20%, 30%, 40%, 50%, 60%, 70). %, 80%, 85%, 90%, 95% and 99%). Purity can be measured by any suitable standard method, for example by column chromatography analysis, polyacrylamide gel electrophoresis analysis or HPLC analysis. The isolated polypeptides described in the present invention can be purified from natural sources and can be produced by recombinant DNA technology or by chemical methods.

A "recombinant" polypeptide refers to a polypeptide produced by recombinant DNA technology, i.e., a polypeptide produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide. A "synthetic" polypeptide refers to a polypeptide prepared by chemical synthesis. The term "recombinant" is used, for example, in connection with a cell, nucleic acid, protein or vector, where the cell, nucleic acid, protein or vector is introduced by the introduction of a heterologous nucleic acid or protein, or is a native nucleic acid or Indicates that the cell has been modified by a change in protein, or that the cell is derived from such modified cell.

"Overexpression" refers to the expression of RNA or polypeptide encoded by a nucleic acid introduced into a host cell, provided that such RNA or polypeptide or protein is normally present in the host cell. Either not, or such RNA or polypeptide is present in the host cell at a level higher than normally expressed from the endogenous gene encoding the RNA or polypeptide. Is it?

The amino acid composition of each of the above-mentioned polypeptides does not interfere with their function, i.e., upregulates the above-mentioned network (eg, increases the activation level of the ApoE / LRP signaling pathway), thereby thereby. It may change without interfering with its ability to inhibit metastasis to multiple organs. For example, the amino acid composition can contain one or more conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Various populations of amino acid residues with similar side chains are defined in the art. These populations include amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamate), amino acids with uncharged polar side chains (eg, eg). Glycin, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains Amino acids (eg, threonine, valine, isoleucine) and amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine) are included. Therefore, it is predicted in one of the above polypeptides (eg, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18). Non-essential amino acid residues are preferably replaced by another amino acid residue from the same side chain population. Alternatively, mutations can be randomly introduced along the whole or part of the sequence, eg, by induction of saturation mutagenesis, and the resulting variants are upregulated through the networks described above or the ApoE / LRP signaling pathway. And can also be screened for their ability to elicit their respective cellular responses to identify mutants that retain activity as described in the Examples below.

Functional equivalents of the polypeptides of the invention represent derivatives of the polypeptide, eg, proteins having one or more point mutations, insertions, deletions, shortenings, fusion proteins, or combinations thereof. The functional equivalent substantially retains the activity of the above-mentioned polypeptides. The isolated polypeptide of the present invention is one sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18. Alternatively, it may contain a functional equivalent or fragment thereof. In general, the functional equivalent is at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18. 75% identity (eg, any number between 75% and 100%, including, for example, 70%, 80%, 85%, 90%, 95% and 99%).

The polypeptide described in the present invention can be obtained as a recombinant polypeptide. To prepare a recombinant polypeptide, another nucleic acid encoding the polypeptide is encoded by a fusion partner (eg, glutathione-s-transferase (GST), 6x-His epitope tag or M13 gene 3 protein). It can be linked to nucleic acids. The resulting fusion nucleic acid expresses in a suitable host cell a fusion protein that can be isolated by methods known in the art. The isolated fusion protein can be further processed, for example, by enzymatic digestion, except for the fusion partner and to obtain the recombinant polypeptide of the invention. Alternatively, the polypeptides of the invention can be chemically synthesized (see, eg, Proteins: Proteins and Molecular Principles) (WH Freeman & Co., NY, 1983). For further guidance, those skilled in the art are described by Ausubel et al. (Currant Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition, 1987 & 1995), Sambrook et al. (Molecular Biology, Molecular Biology).
Spring Harbor Press, Cold Spring Harbor, NY, 1989), and chemical synthesis, Gait, M. et al. J. The edition (oligonucleotide Synthesis, IRL Press, Oxford, 1984) may be examined.

Due to their function as cellular or membrane proteins, DNAJA4, LRP1, LRP8 and LXR can be linked to one or more of the amino acid sequences, including the Cell Permeable Peptide (CPP) sequence and the like. , For example, they can be conjugated or fused to them. In this way, the compositions of the invention as discussed below can include transport enhancers. Cell-penetrating peptides (CPPs) generally consist of less than 30 amino acids and have a net positive charge. Various CPPs translocate in living animal cells in an endocytic or receptor / energy-independent manner. Several classes of CPPs exist with varying origins, from fully protein-derived CPPs to fully synthetic CPPs via chimeric CPPs. Examples of various CPPs are known in this art. See, for example, U.S. Patent Application Publication Nos. 2009099066 and 20100279918. CPPs are known to be able to deliver exogenous proteins into a variety of cells.

All of the naturally occurring, genetically engineered and chemically synthesized forms of the above-mentioned polypeptides can be used to carry out the inventions disclosed therein. Polypeptides obtained by recombinant DNA technology are in naturally occurring forms (eg, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16). And one of SEQ ID NOs: 18) or may have the same amino acid sequence as its functional equivalent. They also include chemically modified forms. Examples of chemically modified polypeptides include conformational changes, polypeptides subject to side chain additions or deletions, and polypeptides to which compounds such as polyethylene glycol are attached. The polypeptide is included in a suitable composition when purified and tested by standard methods, or according to the methods described in Examples below, or other methods known in the art. Can be done.

To express the factors described above, the present invention provides nucleic acids encoding any of the polypeptides described above. Preferably, the nucleotide sequence is isolated and / or purified. Nucleic acid can be a DNA molecule (eg, cDNA or genomic DNA, but not limited to), an RNA molecule (eg, mRNA, but not limited to), or a DNA analog or RNA analog. DNA or RNA analogs can be synthesized from nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded. An "isolated nucleic acid" is a nucleic acid whose structure is not identical to the structure of any naturally occurring nucleic acid or to any fragment of a naturally occurring genomic nucleic acid. Thus, the term includes, for example: (a) a sequence of a portion of a naturally occurring genomic DNA molecule, but in the genome of an organism in which the molecule is naturally present. DNA in which both of the coding sequences located on both sides of such a portion are not flanked; (b) in a vector such that the resulting molecule is not identical to any naturally occurring or genomic DNA. Alternatively, a nucleic acid integrated into the genomic DNA of a prokaryotic or eukaryotic organism; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment brought about by a polymerase chain reaction (PCR), or a restricted fragment; and (d) a hybrid. A gene, a recombinant nucleotide sequence that is part of a gene encoding a fusion protein.

The terms "RNA", the term "RNA molecule" and the term "ribonucleic acid molecule" are used interchangeably herein to indicate a polymer of ribonucleotides. The term "DNA" or the term "DNA molecule" or the term "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized in nature (eg, by DNA replication or DNA transcription, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. The DNA and RNA can be single-stranded (ie, ssRNA and ssDNA, respectively) or multi-stranded (eg, double-stranded, ie, dsRNA and dsDNA, respectively).

The present invention also provides recombinant constructs having one or more of the nucleotide sequences described herein. Examples of such constructs include vectors in which the nucleic acid sequences of the invention are inserted in a forward or reverse orientation (eg, a plasmid or viral vector). In a preferred embodiment, the construct further comprises various regulatory sequences, including a promoter operably linked to the sequence. A large number of suitable vectors and promoters are known to those of skill in the art and are commercially available. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press).

Examples of expression vectors include chromosomal DNA sequences, non-chromosomal DNA sequences and synthetic DNA sequences, such as derivatives of Simian virus 40 (SV40), bacterial plasmids, phage DNA, baculovirus, yeast plasmids, plasmids and phage DNA. Vectors derived from the combination of, viral DNA (eg, vaccinia, adenovirus, chicken pox virus and pseudomad dog disease, etc.) are included. However, any other vector may be used as long as it is replicable and viable in the host. The appropriate nucleic acid sequence may be inserted into the vector by various procedures. In general, the nucleic acid sequence encoding one of the polypeptides described above can be inserted into the appropriate restriction endonuclease site by procedures known in the art. Such procedures and related subcloning procedures are within the scope of those skilled in the art.

The nucleic acid sequence in the expression vector described above is preferably operably linked to a suitable transcriptional control sequence (promoter) for deriving mRNA synthesis. Examples of such promoters are the long terminal (LTR) or SV40 promoter of retrovirus, the lac or trp promoter of Escherichia coli, the PL promoter of phage lambda, and the expression of genes in prokaryotic or eukaryotic cells or viruses. Includes other promoters known to regulate in. The expression vector can also contain a ribosome binding site for initiation of translation and a transcription terminator. The vector may contain suitable sequences for amplifying expression. In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells (eg, eukaryotic cell cultures). For dihydrofolate reductase or neomycin resistance, etc., or tetracycline resistance or ampicillin resistance in Escherichia coli, etc.).

A vector containing the appropriate nucleic acid sequence as described above, as well as the appropriate promoter or control sequence, transforms the appropriate host cell to give the host the polypeptide described above. It can be used to make it possible to express. Such vectors can be used in gene therapy. Examples of suitable expression hosts include bacterial cells (eg, Escherichia coli, genus Streptomyces, Salmonella typhimurium), fungal cells (yeast), insect cells (eg, Spodoptera frugiperda) (Sf9). , Animal cells (eg, CHO, COS and HEK293), adenovirus and plant cells. The choice of an appropriate host is within the skill of one of ordinary skill in the art. In some embodiments, the invention provides a method for producing the above-mentioned polypeptides by transfecting host cells with an expression vector having a nucleotide sequence encoding one of these polypeptides. .. Host cells are then cultured under appropriate conditions to consider expression of the polypeptide.

Reducing the expression or activity level of metastasis-promoting factors As mentioned above, inhibitory agents that reduce the expression or activity level of miR-199a-3p, miR-199a-5p, miR-1908 or CTGF. , Can be used in the treatment of melanoma. The inhibitory agent (ie, inhibitor) can be a nucleic acid, polypeptide, antibody or small molecule compound. In one example, the inhibitor functions at the levels of transcription, mRNA stability, translation, protein stability / degradation, protein modification and protein binding.

Nucleic acid inhibitors can encode small interfering RNAs (eg, RNAi agents) that target one or more of the genes described above (eg, CTGF) and inhibit their expression or activity. The term "RNAi agent" refers to RNA or an analog thereof that has sufficient sequence complementarity to the target RNA to induce RNA interference. Examples also include DNA that can be used to make such RNA. RNA interference (RNAi) refers to a sequence-specific or sequence-selective process in which a target molecule (eg, a target gene, protein or RNA) is downregulated. In general, interfering RNA (“iRNA”) is a short double-stranded interference that can be used to cause catalytic degradation of a particular mRNA and also to reduce or inhibit gene expression. Sex RNA (siRNA), short hairpin RNA (SHRNA) or single-stranded microRNA (miRNA).

The term "short interfering RNA" or the term "siRNA" (which is also known as "small interfering RNA") is about 10 to 50 nucleotides in length, preferably about 15 nucleotides in length. Between nucleotides to 25 nucleotides, more preferably about 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides or 25 nucleotides in length, optionally with strands. For example, an RNA drug (preferably a double-stranded drug) having a protruding end containing one, two or three protruding nucleotides (or nucleotide analogs) can induce or mediate RNA interference. Show what you can do. Naturally occurring siRNAs are generated from longer dsRNA molecules (eg, over 25 nucleotides in length) by cellular RNAi devices (eg, dicers or homologs thereof).

The term "miRNA" or the term "microRNA" is about 10 to 50 nucleotides in length, preferably between about 15 and 25 nucleotides in length, more preferably about 17 nucleotides in length. RNA agents (preferably double-stranded agents) that are 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides or 25 nucleotides can induce or mediate RNA interference. Show what you can do. Naturally occurring miRNAs are generated by dicers from stem-loop precursor RNAs (ie, pre-miRNAs). As used herein, the term "dicer" refers to any dicer ortholog that can process a dicer, as well as a dsRNA structure, into a siRNA, miRNA, siRNA-like molecule or miRNA-like molecule. Or dicer homologs are also included. The term microRNA (or "miRNA") is due to the fact that naturally occurring microRNAs (or "miRNAs") have been found to be expressed in a transient fashion (eg, during development). Based on this, it is used interchangeably with the term "small transient RNA" (or "stRNA").

The term "SHRNA" as used herein refers to an RNA agent having a stem-loop structure comprising a first region and a second region of a complementary sequence, but in this case these. The degree of complementarity and orientation of the regions is sufficient so that base pairing occurs between these regions, the first and second regions are connected by loop regions, and the loops are nucleotides within the loop regions. It results from the absence of base pairing between (or nucleotide analogs).

Within the scope of the present invention is the use of RNAi characterized by the degradation of RNA molecules (eg, intracellular degradation). Degradation is enzymatic RNA-induced silencing complex (RISC)
Catalyzed by. An RNA drug having a sequence that is sufficiently complementary to the target RNA sequence (eg, the CTGF gene described above) to derive RNAi is such that the RNA drug is at least 50% homologous to the target RNA sequence (eg, the CTGF gene described above). For example, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% homology), so that these two hybridize and target. It means that RNA disruption is sufficiently complementary to each other to be induced by RNAi devices (eg, RISC complexes) or RNAi processes. An RNA drug having "a sequence that is sufficiently complementary to the target RNA sequence to derive RNAi" is also sufficient for the RNA drug to induce translational inhibition of the target RNA by an RNAi device or RNAi process. It means having a certain sequence. RNA agents can also have sequences that are sufficiently complementary to the target RNA encoded by the target DNA sequence so that the target DNA sequence is silenced by chromatin. In other words, RNA agents are targeted DNA sequences to induce transcribed gene silencing, eg, by inducing gene expression, eg, structural changes in chromatin, at or near the target DNA sequence. It has a sequence that is sufficient for down-regulation in or near it.

The polynucleotides described above can be delivered using polymeric biodegradable microparticle delivery devices or biodegradable microcapsule delivery devices known in the art. Another way to achieve polynucleotide uptake is to use liposomes, which are prepared by standard methods. Polynucleotides can be integrated alone or with tissue-specific antibodies in these delivery vehicles. Alternatively, a molecular conjugate composed of a plasmid or other vector that binds to poly-L-lysine by electrostatic or covalent force can be prepared. Poly-L-lysine binds to a ligand capable of binding to a receptor on the target cell (Cristiano et al., 1995, J. Mol. Med., 73: 479). Alternatively, tissue-specific targeting can be achieved by the use of tissue-specific transcriptional regulatory elements known in the art. Delivery of naked DNA to intramuscular, intradermal or subcutaneous sites (ie, delivery without a delivery vehicle) is another means of achieving in vivo expression.

SiRNA molecules, miRNA molecules and asRNA (antisense RNA) molecules can be designed by methods widely known in the art. AMBION, Inc. has described siRNA, miRNA and asRNA molecules that have sufficient homology to provide the sequence specificity required to uniquely degrade any RNA. And DHARMACON, Inc. Various programs known in the art can be designed using, including, but not limited to, programs maintained on the website for. Systematic testing of several designed species to optimize such siRNA, miRNA and asRNA sequences can be routinely performed by one of ordinary skill in the art. Considerations when designing short interfering nucleic acid molecules include biophysical, thermodynamic and structural considerations, base preference at specific positions in the sense strand, and homology. Not limited to. These considerations are widely known in the art and provide guidance for designing the RNA molecules described above.

Which antisense polynucleotide (preferably DNA) of the present invention has a base sequence that is complementary or substantially complementary to the base sequence of the gene encoding the components of the network described above. It can also be an antisense polynucleotide such as. The base sequence can be at least about 70%, 80%, 90% or 95% homologous to the complement of the gene encoding the polypeptide. These antisense DNAs can be synthesized using a DNA synthesizer.

The antisense DNA of the present invention may contain altered or modified sugars, bases or linkages. Antisense DNA, as well as the RNAi agents mentioned above, may also be provided in specialized forms, such as liposomes, microspheres, etc., or when applied to gene therapy. Or may be provided in combination with bound components. Such bound components may have polycations (eg, polylysine) that act as charge neutralizers for the phosphate skeleton, or hydrophobicity that enhances interaction with cell membranes or increases nucleic acid uptake. Includes sex components (eg, lipids (eg, phospholipids, cholesterol, etc.)). A preferred example of the lipid to be bound is cholesterol or a derivative thereof (eg, cholesteryl chloroformate, cholic acid, etc.). These components may be attached to the nucleic acid at its 3'end or 5'end, and may also be attached to the nucleic acid via a base, sugar or intermolecular nucleoside linkage. Other components may be capping groups specifically placed at the 3'or 5'end of nucleic acid to prevent degradation by nucleases (eg, exonucleases, RNases, etc.). Such capping groups include, but are not limited to, various hydroxyl protecting groups known in the art, including, but not limited to, glycols such as polyethylene glycol and tetraethylene glycol. The inhibitory effect of antisense DNA can be investigated using the gene expression system of the invention based on cell lines or animals in vivo and in vitro.

The nucleic acids discussed above encoding one or more of the polypeptides described above or RNAi agents can be cloned into vectors for delivery to cells in vitro or in vivo. For in vivo use, delivery can target specific tissues or organs (eg, skin). Targeted delivery involves the use of vectors (eg, organ homing peptides) that are targeted after systemic administration to a particular organ or tissue. For example, the vector can have a covalent conjugate of avidin with a monoclonal antibody against a liver-specific protein.

In some specific embodiments, the invention provides a method for in vivo expression of the above-mentioned metastasis-suppressing factors. In such methods, human or non-human animal cells or human or non-human animal cells whose therapeutic effect requires the nucleic acid sequence encoding any of the above factors to inhibit endothelial recruitment, cancer cell infiltration or metastatic angiogenesis. It will be achieved by introducing it into the organization. Delivery of nucleic acid sequences can be achieved using recombinant expression vectors (eg, chimeric viruses) or colloidal dispersion systems. Preferred for therapeutic delivery of nucleic acid sequences is the use of targeted liposomes.

The various viral vectors available for gene therapy disclosed herein include adenovirus, adeno-related virus (AAV), herpesvirus, vaccinia, or preferably RNA virus (eg, retro). Viruses and lentiviruses, etc.) are included. Preferably, the retroviral vector is a lentivirus or a derivative of a mouse or avian retrovirus. Examples of retrovirus vectors in which only one foreign gene can be inserted are Moloney mouse leukemia virus (MoMuLV), Harvey mouse sarcoma virus (HaMuSV), mouse breast cancer virus (MuMTV) and Rous sarcoma virus (RSV). Includes, but is not limited to. Numerous additional retroviral vectors can incorporate a large number of genes.

All of these vectors are capable of transferring or incorporating genes for selectable markers so that transduced cells can be identified and produced. Retroviral vectors can be targeted-specific, for example by binding sugars, glycolipids or proteins. Preferred targeting is achieved by using target-specific antibodies or hormones that have the receptor at the target. Those skilled in the art recognize that specific polynucleotide sequences can be inserted into the genome of a retrovirus or bound to the envelope of the virus to allow target-specific delivery of the retroviral vector. Will.

Another targeted system for delivering nucleic acids is the colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. The preferred colloidal system of the present invention is a liposome. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated in an aqueous solution in biologically active form and delivered to cells. Various methods for efficient introgression using liposome vehicles are known in the art. The composition of liposomes is usually a combination of phospholipids in combination with steroids, in particular a combination of phospholipids in combination with cholesterol. Other phospholipids or other lipids may also be used. The physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations.

Examples of lipids useful in liposome fabrication include sphingolipids, cerebrosides and gangliosides such as phosphatidyl compounds such as phosphatidylglycerols, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. Targeting of liposomes is also possible based on, for example, organ specificity, cell specificity and organelle specificity and is known in the art.

When used in vivo, it is desirable to use a reversible delivery-expression system. To achieve such an end, Cre-loxP or FLP / FRT or other similar systems can be used for reversible delivery-expression of one or more of the nucleic acids. See International Publication WO2005 / 112620, International Publication WO2005 / 039643, US Patent Application Publication Nos. 20050130919, 20030022375, 2002202218, 20030027335 and 20040216178. In particular, the reversible delivery-expression system described in US Patent Application Publication No. 200824990 can be used to provide selective or emergency interruption.

In another example, the inhibitory agent described above can be a polypeptide or protein complex, such as an antibody. The term "antibody" refers to an immunoglobulin molecule, or immunologically active portion thereof, i.e., an antigen binding moiety. Examples include proteins having at least one or two heavy (H) chain variable regions ( _VH ) and at least one or two light (L) chain variable regions ( _VL ). Not limited to. The _VH and _VL regions are also hypervariable regions (which are called "complementarity determining regions"("CDRs"), which are interspersed with more conserved regions (this is called the "framework regions" (FR)). ) Can be subdivided into). As used herein, the term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by an immunoglobulin gene. The recognized human immunoglobulin genes include kappa constant region genes, lambda constant region genes, alpha constant region genes (IgA1 and IgA2), gamma constant region genes (IgG1, IgG2, IgG3 and IgG4), delta constant region genes, It contains the Epsilon constant region gene and the Mu constant region gene, as well as a myriad of immunoglobulin variable region genes.

The term "antigen binding portion" (or "antibody moiety") of an antibody refers to one or more fragments of an antibody that retains the ability to specifically bind to an antigen (eg, LRP1, LRP8 and CTGF). It has been shown that the antigen-binding function of an antibody can be accomplished by a fragment of a full-length antibody. Examples of binding fragments that fall within the term "antigen binding portion" of an antibody include (i) Fab fragments, i.e., one consisting of a _VL domain, a _VF domain, a _CL domain and a _CH1 domain. Valuable Fragments; (iii) F (ab') ₂ Fragments, ie, Bivalent Fragments Containing Two Fab Fragments Linked by Disulfide Bridges in the Hinge Regions; (iii) Fd Fragments Consisting of _VH Domains and _CH1 Domains; (Iv) Fv fragment consisting of _VL domain and _VH domain of only one arm of antibody; (v) dAb fragment consisting of _VH domain (Ward et al. (1989), Nature, 341: 544-546); Also included are (vi) isolated complementarity determining regions (CDRs). Moreover, although the two domains of the Fv fragment, namely _VL and _VE , are encoded by separate genes, they are monovalent molecules in which the _VL and _VH regions are paired. It can be linked using a recombinant method (eg, Bird) by a synthetic linker that allows it to be made as a single protein chain (known as a single chain Fv (scFv)). Et al. (1988), Science, 242: 423-426, and Huston et al. (1988), Proc. Natl. Acad. Sci. USA, 85: 5879-5883). Such single chain antibodies are also intended to be included within the term "antigen binding portion" of the antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for usefulness in the same manner as intact antibodies are screened.

Antibodies that specifically bind to one of the above-mentioned target proteins (eg, CTGF) can be made using methods known in the art. This antibody can be a polyclonal or monoclonal antibody. In one embodiment, the antibody can be produced recombinantly, for example by phage display or by combinatorial methods. In another embodiment, the antibody is a fully human antibody (eg, an antibody produced in a mouse genetically engineered to produce the antibody from a human immunoglobulin sequence), a humanized antibody, or a non-human antibody. For example, rodents (mouse or rat), goats, primates (eg, monkeys, but not limited to), rabbit or camel antibodies (but not limited to these). Examples of various methods for producing humanized forms of antibodies include CDR grafting (Queen et al., US Pat. No. 5,585,089; Richmann et al., Nature, 332: 323 (1988)), chain shuffling. Rings (US Pat. No. 5,565,332) and veneering or surface reconstruction (European Patent No. 592,106; European Patent No. 519,596; Padlan, Protein Immunology, 28 ( 415): 489-498 (1991); Studnicka et al., Protein Engineering, 7 (6): 805-814 (1994); Roguska et al., PNAS, 91: 969-973 (1994)), but limited to these. Not done. Examples of various methods for producing fully human antibodies are to generate antibodies from mice capable of expressing human immunoglobulin genes, and to create and screen human immunoglobulin gene libraries. Includes, but is not limited to, the use of phage display techniques.

An "isolated antibody" is intended to indicate an antibody that is substantially free of other antibodies with different antigen specificities (eg, an isolated antibody that specifically binds to CTGF). It is substantially free of antibodies that specifically bind to an antigen other than such an antigen). Moreover, the isolated antibody may be substantially free of other cellular and / or chemicals.

The term "monoclonal antibody" or the term "monoclonal antibody composition", as used herein, refers to a preparation of an antibody molecule having a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

As used herein, the term "human antibody" is intended to include antibodies in which both framework and CDR regions have variable regions derived from the immunoglobulin sequences of the human germline. Furthermore, if the antibody contains a constant region, this constant region is also derived from the immunoglobulin sequence of the human germline. The human antibodies of the invention are mutations introduced by amino acid residues not encoded by the immunoglobulin sequence of the human germline (eg, by random or site-directed mutagenesis in vitro, or by somatic mutations in vivo. ) May be included. However, as used herein, the term "human antibody" is a CDR sequence derived from the germline of another mammalian species (eg, mouse, etc.) grafted onto a human framework sequence. It is not intended to include antibodies.

The term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity in which both the framework and CDR regions have variable regions derived from the immunoglobulin sequences of the human germline. In one embodiment, a human monoclonal antibody is a B cell obtained from a transgenic non-human animal (eg, recombinant mouse) having a genome comprising a human heavy chain transgene and a light chain transgene. Is produced by a hybridoma containing, fused to immortalized cells.

The term "recombinant human antibody" as used herein includes all human antibodies prepared, expressed, produced or isolated by recombinant means, eg, the following antibodies. Such as: (a) Antibodies isolated from animals (eg, mice) that are recombinant or chromosomally recombinant for human immunoglobulin genes, or from hybridomas prepared from such animals (which are described below). (B) Antibodies isolated from host cells transformed to express human antibodies, such as antibodies isolated from transfectomas; (c) recombinant combinatorial human antibodies. Antibodies isolated from the library; and (d) Antibodies that are prepared, expressed, produced or isolated by any other means involving splicing the human immunoglobulin gene sequence onto other DNA sequences. .. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from the immunoglobulin sequences of the human germline. However, in some particular embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when animals that are recombinant for human Ig sequences are used). Thus, while the amino acid sequences of the _VH and _VL regions of a recombinant antibody can be derived from or associated with the _VH and _VL sequences of human germline, of course, in vivo. Is a sequence that may not be present in the germline repertoire of human antibodies.

As used herein, "isotype" refers to an antibody class encoded by a heavy chain constant region gene (eg, IgM or IgG1). The expressions "antibody that recognizes an antigen" and the expression "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen". As used herein, the term "high affinity" means that for an IgG antibody, the antibody has a KD of ^10-7 _M or less, preferably a KD of ^10-8 _M or less for the target antigen. It is shown to have, more preferably to have a KD of ^10-9 _M or less, and even more preferably to have a KD of ^10-10 _M or less. However, "high affinity" binding can be different for other antibody isotypes. For example, a "high affinity" binding for the IgM isotype indicates that the antibody has a KD of ^10-7 _M or less, more preferably a KD of ^10-8 _M or less.

In one example, the composition contains a monoclonal antibody that neutralizes CTGF. In one embodiment, the antibody is a fully human antibody, a humanized antibody, or a non-human antibody such as a rodent (mouse or rat), goat, primate (eg, monkey, but not limited to). It can be a rabbit or camel antibody, but not limited to these. In one embodiment, one or more amino acids of this monoclonal monoclonal antibody may be substituted to alter its various physical properties. These properties include, but are not limited to, binding specificity, binding affinity, immunogenicity and antibody isotypes. Pharmaceutical compositions containing the fully human or humanized form of the antibodies can be used to treat melanoma or to inhibit endothelial recruitment, cancer cell infiltration or metastatic angiogenesis. can.

As used herein, "subject" refers to humans and non-human animals. Examples of non-human animals include all vertebrates such as mammals such as non-human mammals, non-human primates (particularly higher primates), dogs, rodents (eg mice or rats). Includes guinea pigs, cats and rabbits, as well as non-mammals such as birds, amphibians, reptiles and the like. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or an animal suitable as a disease model. Targets to be treated for a disorder can be identified by standard diagnostic techniques for that disorder. If necessary, the subject should have the miR-199a-3p, miR-199a-5p, miR-1908 and CTGF described above prior to treatment by methods known in the art or those described above. One or more mutations, expression levels or activity levels of can be investigated. If the subject has a particular mutation in the gene, or if the gene expression or activity level is higher than, for example, in the sample obtained from the subject, in the sample obtained from a normal subject. , The subject is a candidate for the treatment of the present invention.

To confirm inhibition or treatment, evaluation and / or confirmation of endothelial mobilization or resulting inhibition of angioplasty prior to and / or after the administration step using techniques known in the art. Can be done. Exemplary techniques include angiography or angiography, which are medical imaging techniques used to visualize the interior or lumen of blood vessels and organs in the body, generally radiation-impermeable contrast agents. It can be done by injecting into a blood vessel and imaging using X-ray based techniques, for example, using fluoroscopy.

"Treatment" or "treatment," as used herein, refers to a subject with a disability as the disorder, symptoms of the disorder, a disease state secondary to the disorder, or the disorder. Indicates that a compound or drug is administered with the purpose of treating, alleviating, alleviating, curing, delaying, preventing, or ameliorating the predisposition to. "Effective amount" or "therapeutic effective amount" refers to the amount of compound or agent that can produce medically desirable results in a treated subject. Treatment methods can be performed in vivo or in vivo, either alone or in combination with other drugs or therapies. A therapeutically effective amount can be administered in a single or multiple doses, application or dosing, and the therapeutically effective amount is not intended to be limited to a particular formulation or route of administration.

The expression "effective amount", as used herein, refers to a sufficient amount of a compound of the invention to exhibit the desired therapeutic effect. The exact amount required will vary from subject to subject depending on the subject's species, age and general condition, as well as specific therapeutic agents and the like. The compounds of the present invention are preferably formulated in a dosage unit form for ease of administration and uniformity of dosage. The expression "dosing unit form", as used herein, refers to a physically separate unit of therapeutic agent that is appropriate for the patient to be treated. However, it will be appreciated that the total daily use of the compounds and compositions of the invention will be determined by the attending physician within reasonable medical judgment. The specific therapeutically effective dose level for a particular patient or organism of any patient or organism is the disorder being treated and the severity of the disorder; the anticancer activity of the specific compound used. Specific composition used; patient's age, weight, general health, gender and diet; time of administration, route and excretion rate of specific compound used; duration of treatment; specific used Drugs used in combination with or at the same time as compounds; as well as a variety of factors, including similar factors widely known in the medical technology field.

Therapeutic agents can be administered alone in vivo or in vivo, or in combination with other drugs or therapies, ie, co-administered in cocktail therapy. As used herein, the term "co-administration" or the term "co-administered" indicates that at least two agents or therapies are to be administered. For example, in the treatment of tumors, especially in the treatment of angiogenic malignancies, the agents can be used alone or, for example, chemotherapeutic agents, radiotherapy agents, apoptosis agents, anti-angiogenic agents and / or immunotoxins or coagligants ( It can be used in combination with (coaguligand). In some embodiments, co-administration of two or more agents / therapies is simultaneous. In other embodiments, the first agent / treatment is applied prior to the second agent / treatment. One of ordinary skill in the art understands that the formulations and / or routes of administration of the various agents / treatments used may differ.

In an in vivo effort, a compound or agent is administered to a subject. Generally, the compound is suspended in a pharmaceutically acceptable carrier (eg, but not limited to physiological saline) and administered orally or by intravenous infusion, or subcutaneously, intramuscularly, arachnoid. It is injected or implanted intrathecal, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally or intrapulmonary.

The required dosage depends on the choice of route of administration; the nature of the formulation; the nature of the patient's illness; the size, weight, surface area, age and gender of the subject; other drugs being administered; and the judgment of the attending physician. .. Suitable dosages are in the range of 0.01 mg / kg to 100 mg / kg. Changes in the required dosage are expected taking into account the different compounds available and the different efficiencies of the different routes of administration. For example, oral administration is i. v. It would be expected to require higher dosages than administration by injection. Changes in these dosage levels can be adjusted using standard empirical practices for optimization as well understood in the art. Encapsulation of a compound in a suitable delivery vehicle (eg, a polymer microparticle or an implantable device) can increase delivery efficiency, especially for oral delivery.

Compositions Within the scope of the invention are compositions containing a suitable carrier and one or more of the therapeutic agents described above. The composition is a pharmaceutical composition containing a pharmaceutically acceptable carrier, a dietary composition containing a suitable carrier acceptable for diet, or a cosmetic composition containing a carrier acceptable for cosmetics. It is possible.

The term "pharmaceutical composition" refers to a combination of an active agent and a carrier (inactive or active) that makes the composition particularly suitable for diagnostic or therapeutic use in vivo or in vivo. A "pharmaceutically acceptable carrier" does not cause unwanted physiological effects after administration or application to a subject. The carrier in the pharmaceutical composition must also be "acceptable" in the sense that it is compatible with the active ingredient and is capable of stabilizing the active ingredient. One or more solubilizers can be utilized as pharmaceutical carriers for delivering the active compound. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives and diluents for achieving compositions that can be used as dosage forms. .. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D & C Yellow # 10.

As used herein, the term "pharmaceutically acceptable salt" is used, within reasonable medical judgment, without excessive toxicity, irritation and allergic responses, etc., in humans and inferior. Such salts are suitable for use in contact with animal tissues and that correspond to a reasonable benefit / risk ratio. Various pharmaceutically acceptable salts of amines, carboxylic acids and other types of compounds are widely known in the art. For example, S. M. Berge et al. Have published various pharmaceutically acceptable salts in J. Mol. It is described in detail in Pharmaceutical Sciences, 66: 1-19 (1977) (which is incorporated herein by reference). Such salts can be prepared in situ during the final isolation and purification period of the compounds of the invention, or, as generally described below, free basic functional groups or It can be prepared separately by reacting the free acid functional group with a suitable reagent. For example, a free base functional group can be reacted with a suitable acid. Further, when the compound of the present invention has an acidic component, suitable pharmaceutically acceptable salts thereof include metal salts, for example, alkali metal salts (eg, sodium salt or potassium salt) and alkaline earth metal salts. (For example, calcium salt or magnesium salt) may be included. Examples of pharmaceutically acceptable non-toxic acid addition salts are inorganic acids (eg, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, etc.) or organic acids (eg, acetic acid, oxalic acid, maleine). Formed by using a salt of an amino group formed by an acid, tartaric acid, citric acid, succinic acid or malonic acid, or by using other methods used in the art (eg ion exchange). Amino group salts can be mentioned. Other pharmaceutically acceptable salts include adipates, alginates, ascorbics, asparaginates, benzenesulfonates, benzoates, bicarbonates, borates, butyrate, succinates. , Drosophila sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate Salt, heptanoate, hexanenate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonic acid Salt, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate , Phosphate, picphosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate and valerate, etc. Is included. Typical alkaline salts or alkaline earth metal salts include sodium, lithium, potassium, calcium and magnesium. Further pharmaceutically acceptable salts include non-toxic ammonium cations, quaternary ammonium cations and amine cations formed using counterions, where appropriate (eg, halides, hydroxides, etc.). Carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates, etc.).

As described above, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier, in which case the pharmaceutically acceptable carrier, when used herein. Any solvent, diluent or other liquid vehicle, dispersion or suspension aid, surfactant, isotonic, thickener or emulsifier, storage to suit the particular dosage form desired. Includes agents, solid binders and lubricants. Remington's Pharmaceutical Sciences, 16th Edition, E.I. W. Martin (Mack)
Publishing Co., Ltd. , Easton, Pa. , 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation. Whatever carrier medium, the conventional carrier medium, eg, interacts with some other component of the pharmaceutical composition in a detrimental manner, for example, with some undesired biological effect or otherwise. Its use is intended to be within the scope of the invention, except as long as it is incompatible with the compounds of the invention. Some examples of substances that may serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and Potato starch and the like; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacant; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanuts. Oils, cottonseed oil, benivana oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; natural and synthetic phospholipids such as , Soybean and egg yolk phosphatid, lecithin, hydrided soybean lecithin, dimyristoyl lecithin, dipalmitoyle lecithin, distearoyl lecithin, dioleoyl lecithin, hydroxylated lecithin, lysophosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine Diastealoyl phosphatidylethanolamine (DSPE) and pegated esters thereof (eg, DSPE-PEG750 and DSPE-PEG2000, etc.), phosphatidyl acid, phosphatidylglycerol and phosphatidylserine and the like. Preferred commercial standard products of lecithin include those available under the trade names of Phosal® or Phosphoripon®, Phosal 53 MCT, Phosal 50 PG, Phosal 75 SA, Phosphoripon 90H, Phosphol. , And Phosphoripon 90 NG; soybean phosphatidylcholine (SoyPC) and DSPE-PEG2000 are particularly preferred; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic physiological Salt solution; Ringer's solution; Ethyl alcohol and phosphate buffer, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, mold release agents, coatings. Agents, sweeteners, flavors and fragrances, preservatives and antioxidants can also be present in the composition at the discretion of the prescriber.

The composition can be used to treat melanoma, or any other disease or condition described herein, in any of the forms described above. Effective amount indicates the amount of active compound / drug required to deliver a therapeutic effect to the treated subject. Effective doses vary depending on the type of disease being treated, route of administration, excipient usage, and potential for concomitant use with other therapeutic treatments, as will be recognized by those of skill in the art. Will do.

The pharmaceutical composition of the present invention can be administered by parenteral administration, oral administration, nasal administration, rectal administration, local administration or oral administration. The term "parenteral" as used herein, subcutaneous injection, intracutaneous injection, intravenous injection, intramuscular injection, intra-articular injection, intraarterial injection, intracapsular injection, intrathoracic injection, Subepithelial injection, intralesional or intracranial injection, as well as any injection technique, show suitable injection techniques.

The sterile injectable composition can be a solution or suspension in a non-toxic parenteral acceptable diluent or solvent. Such solutions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, fixed oils have traditionally been used as solvents or suspension media (eg, synthesized monoglycerides or diglycerides). Fatty acids (eg, but not limited to oleic acid) such as naturally pharmaceutically acceptable oils, such as, but not limited to, olive oil or castor oil, as their polyoxyethylated forms are useful in the preparation of injectables. Etc.) and their glyceride derivatives are useful in the preparation of injections. These oil solutions or oil suspensions can also contain long chain alcohol diluents or dispersants, such as, but not limited to, carboxymethyl cellulose or similar dispersants. Other commonly used surfactants such as, but not limited to, various Tweens or various Spans or other similar emulsifiers or bioavailability enhancers (these are pharmaceutically acceptable solid forms). , Which are commonly used in the manufacture of liquid forms or other dosage forms) can also be used for compounding purposes.

Compositions for oral administration, whatever dosage form, include capsules, tablets, emulsions, as well as aqueous suspensions, aqueous dispersions and aqueous solutions, which are orally acceptable dosage forms. It is possible. In the case of tablets, commonly used carriers include, but are not limited to, lactose and corn starch. Lubricants, such as, but not limited to, magnesium stearate, are also typically added. Useful diluents for oral administration in capsule form include, but are not limited to, lactose and dried corn starch. When an aqueous suspension or emulsion is orally administered, the active ingredient can be suspended or dissolved in an oil phase combined with an emulsifier or suspending agent, if desired, a particular sweetness. Agents, flavors or colorants can be added.

Pharmaceutical compositions for topical administration according to the described invention can be formulated as solutions, ointments, creams, suspensions, lotions, powders, pastes, gels, sprays, aerosols or oils. Alternatively, the topical formulation can be in the form of a patch or bandage impregnated with the active ingredient (s), optionally containing one or more excipients or diluents. Is. In some preferred embodiments, the topical formulation comprises a substance that will enhance the absorption or penetration of the active agent through the skin or other affected area.

The topical composition contains a safe and effective amount of a dermatologically acceptable carrier suitable for application to the skin. "Cosmetically acceptable" or "dermatologically acceptable" compositions or ingredients are for use in contact with human skin without excessive toxicity, incompatibility, instability and allergic responses, etc. Indicates a composition or ingredient suitable for. The carrier allows the active agent and the ingredients used as needed to be delivered to the skin in appropriate concentrations. Accordingly, the carrier may be a diluent, dispersant or agent to ensure that the active substance is applied to the selected target at the appropriate concentration and is evenly distributed over the selected target. It can act as a solvent or the like. The carrier can be solid, semi-solid or liquid. The carrier can be in the form of a lotion, cream or gel, in particular a form having sufficient thickness or yield point to prevent the active material from settling. The carrier can be inert or have dermatological benefits. The carrier must be physically and chemically compatible with the active ingredients described herein and also excessively compromise the stability, efficacy or other benefits of use associated with the composition. Must not be.

Combination Therapy In some embodiments, the pharmaceutical composition may further comprise additional compounds with antiproliferative activity. Additional compounds with antiproliferative activity can be selected from a group of antiproliferative agents, including the compounds shown in Table 2.

Whether the compounds and pharmaceutical compositions of the invention can be formulated and used in combination therapy, i.e., can the compounds and pharmaceutical compositions be formulated with one or more other desired therapeutic agents or medical techniques. It will also be appreciated that, or may be administered simultaneously with, or prior to, or subsequent to, one or more other desired therapeutic agents or medical procedures. The particular combination of treatments (therapeutic agents or techniques) for use in the combination regimen may take into account the suitability of the desired therapeutic agent and / or method, as well as the desired therapeutic effect to be achieved. There will be. It is also understood that the treatment used may achieve the desired effect for the same disorder, or a different effect (eg, suppression of any adverse effects). Will.

By "anti-proliferative agents", any of which may be used in combination with an LXR agonist to treat the medical conditions listed herein, including such anti-proliferative agents listed in Table 2. , Any antiproliferative agent is intended. Antiproliferative agents also include organic platinum derivatives, naphthoquinone and benzoquinone derivatives, chrysophanoic acid and anthraquinone derivatives thereof.

Methods for Diagnosis and Prognosis The genes can be used in determining whether a subject has or is at risk of having metastatic melanoma. Alternatively, the gene can be used to determine the prognosis of such disorders in a subject.

Diagnostic Methods In one aspect, the invention relates to whether a subject has metastatic melanoma or other disease characterized by endothelial recruitment, cancer cell infiltration or metastatic angiogenesis, or endothelial recruitment, cancer cell infiltration or It provides qualitative and quantitative information for determining whether a person is susceptible to metastatic melanoma or other diseases characterized by metastatic angiogenesis. Subjects with or prone to such disorders are the expression levels, expression patterns or expressions of the genes or products thereof (mRNA, microRNA or polypeptide) in test samples obtained from the subjects. It can be determined based on the profile. In other words, such products can be used as markers to indicate the presence or absence of a disorder. The diagnostic and prognostic assays of the invention include various methods for assessing the expression levels of such products. By these methods, the failure can be detected. For example, a relative increase in the expression level of one or more promoters (ie, miR-199a-3p, miR-199a-5p, miR-1908 and CTGF) implies the presence of a disorder. Conversely, levels or absent below expression imply no impairment.

The presence, level or absence of an mRNA product, microRNA product or polypeptide product in a test sample can be obtained from the test subject, and the test sample can be a compound or agent (eg, a compound or agent capable of detecting nucleic acid). It can be evaluated by contacting it with an RNA probe or DNA probe) or with a compound or drug capable of detecting the polypeptide. "Test samples" include tissues, cells and biological fluids isolated from the subject, as well as tissues, cells and fluids present within the subject's body. The level of expression of the gene of interest can be measured in a number of ways, including measuring the RNA encoded by this gene.

The expressed RNA sample can be isolated from the biological sample using any of several well-known procedures. For example, a biological sample can be lysed in a guanidium-based lysis buffer, which optionally contains additional components to stabilize RNA. In some embodiments, the lysis buffer can contain purified RNA as a control for monitoring the recovery and stability of RNA from cell culture. Examples of such purified RNA templates include kanamycin-positive control RNA from PROMEGA (Madison, WI) and 7.5 kb of poly (A) tailed RNA from LIFE TECHNOLOGIES (Rockville, MD). Is done. The lysate may be used immediately or stored frozen at, for example, −80 ° C.

Optionally, total RNA is cytolyzed (or) using silica-based isolation in a 96-well format capable of adapting to automation, eg, on a RNEASY purification platform (QIAGEN, Inc., Valencia, CA). It can be purified from other types of samples). Other RNA isolation methods are intended (eg, extraction with silica-coated beads or guanidium). Further methods for the isolation and preparation of RNA can be devised by those of skill in the art.

The method of the invention can be performed using crude samples (eg, blood, serum, plasma or cytolysates), which eliminates the need to isolate RNA. R
NAse inhibitors are optionally added to the crude sample. When using crude cell lysates, it should be noted that genomic DNA can provide one or more copies of the target sequence (eg, a gene) depending on the sample. Signals originating from genomic DNA may not be significant in situations where the target sequence is derived from one or more highly expressed genes. However, for genes expressed at low levels, the background can be removed by treating the sample with a DNAse, or targeting the splice junction for subsequent priming of the cDNA or amplification product. It can be removed by using a primer.

The cellular levels of RNA corresponding to the gene can be determined both in vitro and in vitro. RNA isolated from the test sample can be used in hybridization or amplification assays that include Southern or Northern analysis, PCR analysis and probe arrays. The preferred diagnostic method for detecting RNA levels involves contacting the isolated RNA with a nucleic acid probe that can hybridize to the RNA encoded by the gene. The probe is a full-length nucleic acid or a portion thereof (eg, an oligonucleotide that is at least 10 nucleotides in length and is sufficient to specifically hybridize to RNA under stringent conditions). It is possible.

In one form, RNA (or cDNA prepared from RNA) is immobilized on the surface and, for example, the isolated RNA is run on an agarose gel and the RNA is transferred from the gel to membrane (eg, nitrocellulose, etc.). ) Is contacted with the probe. In another form, the probe is surface-immobilized and RNA (or cDNA) is contacted with the probe, for example, in a gene chip array. One of skill in the art can adapt known RNA detection methods to detect RNA levels.

Levels of RNA (or cDNA prepared from RNA) in a sample encoded by the gene to be tested, using nucleic acid amplification, eg, standard PCR (US Pat. No. 4,683,202). ), RT-PCR (Bustin S., J Mol Endocrinol, 25: 169-93, 2000), Quantitative PCR (Ong Y. et al., Hematurology, 7: 59-67, 2002), Real-time PCR (Ginsinger D., Exp Hematol, 30: 503-12, 2002) and in situ PCR (Thaker V., Methods Mol Biol, 115: 379-402, 1999), or any other nucleic acid amplification method, followed by amplified molecules. Can be evaluated by detecting using techniques known in the art. In another embodiment, the method of the invention further contacts the control sample with a compound or agent capable of detecting the RNA of the gene, and the presence of the RNA in the control sample is the RNA in the test sample. Including comparing with the existence of.

The above methods and markers can be used to assess a subject's risk of developing melanoma. Specifically, the invention can be applied to subjects in high-risk cohorts who already have certain risks so as to gain essential insights into early detection. Changes in the level of miR gene products associated with melanoma prior to the development of the transformed phenotype or neobiological phenotype in the cells of interest, or the transformed phenotype or neoplasia in the cells of interest. It can be detected early in the development of the biological phenotype. Accordingly, the invention is also a method for screening subjects at risk of developing melanoma, the level of at least one gene product associated with melanoma, or a combination of gene products associated with melanoma. Methods are provided that include assessing levels in biological samples obtained from the skin of interest. It shows that a subject is at risk of developing melanoma due to changes in the level of the gene product or combination of gene products in the biological sample when compared to the level of the corresponding gene product in the control sample. Is done. Biological samples used for such screening may include skin tissue that is either normal or suspected to be cancerous. Subjects with changes in the level of one or more gene products associated with melanoma are candidates for further monitoring and testing. Such further testing can include histological examination of tissue samples, or other techniques within the scope of one of ordinary skill in the art.

As used herein, the term "diagnosis" means detecting a disease or disorder, or determining the stage or extent of a disease or disorder. Diagnosis of a disease or disorder is usually based on assessing one or more factors and / or symptoms that imply a disease. That is, the diagnosis can be based on the presence, absence or amount of factors implying the presence or absence of a disease or condition. Each factor or symptom that is considered to be implied to diagnose a particular disease need not be exclusively associated with that particular disease. That is, there may be a differential diagnosis that can be inferred from diagnostic factors or symptoms. Similarly, factors or symptoms suggestive of a particular disease may be present in individuals who do not have that particular disease. These diagnostic methods may be used independently or in combination with other diagnostic and / or staging methods known in the medical art for a particular disease or disorder (eg, melanoma). There is.

Prognosis Method The diagnostic methods described above can identify subjects with or at risk of developing melanoma. In addition, changes in the expression levels and / or expression trends of the above-mentioned genes in biological samples, eg, in peripheral blood samples, can provide early signs of recovery or lack thereof. For example, further increases (or decreases) or persistently altered gene expression levels of the promoter gene (or inhibitor gene) indicate a poor prognosis, ie, lack of improvement or poor health. Therefore, these genes can be used to assess recovery after treatment for melanoma. Analysis of this selected set of genes or a subset thereof indicates the outcome of the condition.

The prognosis assays described herein include drugs (eg, agonists, antagonists, peptide mimetics, etc.) for a subject to treat melanoma or other disorders associated with endothelial recruitment, cancer cell infiltration or metastatic angiogenesis. It can be used to determine if a protein, peptide, nucleic acid, small molecule or other drug candidate) is suitable for administration. For example, such an assay can be used to determine if a subject can be administered a chemotherapeutic agent.

Therefore, similarly provided by the present invention is a method of monitoring a subject for treatment for cell proliferation disorders. For this purpose, the gene expression levels of the genes disclosed herein are tested from a subject before, during, or after treatment. You can ask for a sample. The magnitude of change at those levels, as compared to baseline levels, is then evaluated. The reduced expression of the above-mentioned promoter genes (miR-199a-3p, miR-199a-5p, miR-1908 and CTGF) after treatment indicates that the subject can be further treated by the same treatment. There is. Similarly, augmentation with inhibitors (DNAJA4, ApoE, LRP1 and LRP8) also indicates that the subject can be further treated by the same treatment. Conversely, further increases or sustained high expression levels of one or more promoter genes indicate a lack of improvement or diminished health.

The information obtained from performing the above assay can be used to determine the prognosis of a disease and other adverse conditions that affect the health of an individual subject. It is useful in identifying progression and in clinical management of diseases and other adverse conditions that affect the health of individual subjects. In a preferred embodiment, the aforementioned diagnostic assay is characterized by endothelial recruitment, cancer cell infiltration or metastatic angiogenesis in determining the prognosis of melanoma and other conditions characterized by endothelial recruitment, cancer cell infiltration or metastatic angiogenesis. It provides useful information in identifying the progression of melanoma and other conditions, and in the clinical management of melanoma and other conditions characterized by cancer cell infiltration or metastatic angiogenesis. This information helps clinicians more specifically in designing chemotherapeutic regimens or other treatment regimens to eradicate such conditions from the affected body, i.e., from the human body. ..

The term "prognosis", as used herein, refers to a predictive course and outcome of a clinical condition or disease. Prognosis is usually done by assessing the factors or symptoms of the disease that imply a favorable or unfavorable course or outcome of the disease. The expression "determining prognosis", as used herein, refers to a process by which one of ordinary skill in the art can predict the course or outcome of a condition in a patient. The term "prognosis" does not indicate that the course or outcome of the condition can be predicted with 100% accuracy, and instead, one of ordinary skill in the art will have the term "prognosis" with a particular course or outcome. You will understand that it shows an increased probability. That is, one will understand that the course or outcome is more likely to occur in a patient with a given condition when compared to such individuals who do not exhibit the condition.

The terms "favorable prognosis" and the term "positive prognosis" or the terms "unfavorable prognosis" and the term "negative prognosis", as used herein, are the likely course of a condition or disease and / or. A relative term for predicting a promising outcome. A favorable or positive prognosis predicts a better outcome for the condition than an unfavorable or negative prognosis. In a general sense, a "favorable prognosis" is an outcome that is relatively better than many other possible prognosis that may be associated with a particular condition, whereas an unfavorable prognosis is. Predicts a relatively worse outcome than many other possible prognosis that may be associated with a particular condition. Typical examples of favorable or positive prognosis include better than average cure rate, lower tendency for metastasis, longer life expectancy than expected, and differentiation of benign processes from cancerous processes. included. For example, one positive prognosis is a prognosis in which a patient has a 50% chance of being cured of a particular cancer after treatment, while the average patient with the same cancer has only a 25% chance of being cured. ..

The terms "determine", term "measure", term "evaluate" and term "assay" are used interchangeably and include both quantitative and qualitative measurements, as well as characteristics, traits or characteristics. Includes determining if it exists. Evaluation may be relative or absolute. "Evaluating the presence of a target" involves determining the amount of target that is present, as well as determining whether or not the target is present.

Arrays Also provided in the present invention are biochips or arrays. The biochip / array may contain a concatenated solid or semi-solid substrate with the probes described herein or with multiple probes. The probe may be able to hybridize to the target sequence under stringent hybridization conditions. The probes may be linked at a spatially defined address on the substrate. Two or more probes may be used per target sequence, provided that either overlapping probes or probes for different parts of a particular target sequence are used. The probe may be able to hybridize to the target sequence associated with only one disorder recognized by those of skill in the art. The probe may either be synthesized first and then linked to the biochip, or it may be synthesized directly on the biochip.

When "linked" or "immobilized" is used herein to refer to nucleic acids (eg, probes) and solid supports, the binding between the probe and the solid support Means sufficient to be stable under conditions of binding, washing, analysis and removal. The bond may be covalent or non-covalent. Covalent bonds may form directly between the probe and the solid support, or by a cross-linking agent, or specific reactants on either the solid support or the probe, or both. May be formed by inclusion in the molecule of. The non-covalent bond may be one or more of electrostatic, hydrophilic and hydrophobic interactions. Included in non-covalent binding is the covalent binding of a molecule such as streptavidin to the support and the non-covalent binding of the biotinylated probe to this streptavidin. Immobilization may also involve a combination of covalent and non-covalent interactions.

The solid substrate can be modified to contain separate individual sites suitable for probe ligation or association, and can be a material that accepts at least one detection method. Examples of such substrates include glass and modified glass or functionalized glass, plastics (including acrylics, polystyrene, and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, TeflonJ, etc.), polysaccharides, and the like. Includes nylon or polystyrene, resins, silica or silica-based materials (including silicon and modified silicon), carbon, metals, inorganic glass and plastic. The substrate may allow optical detection without producing noticeable fluorescence.

The substrate can be planar, but other shapes of the substrate may be used as well. For example, the probe may be placed on the inner surface of the tube for flow-through sample analysis to minimize sample volume. Similarly, the substrate may be flexible, such as a soft foam, including closed cell foam made from a particular plastic.

Arrays / biochips and probes may be derivatized by chemical functional groups for the subsequent linkage of these two. For example, a biochip may be derivatized with a chemical functional group containing, but not limited to, an amino group, a carboxyl group, an oxo group or a thiol group. When using these functional groups, the probe may be linked using the functional group on the probe either directly or indirectly using a linker. The probe may be linked to a solid support by either a 5'-terminal nucleotide or a 3'-terminal nucleotide, or via an internal nucleotide. The probe may also be attached to a solid support by a non-covalent bond. For example, a biotinylated oligonucleotide can be made, in which case the biotinylated oligonucleotide may be covalently attached to a surface coated with streptavidin, thereby resulting in ligation. .. Alternatively, the probe may be synthesized at the surface using techniques such as photopolymerization and photolithography. A detailed discussion of various methods for linking nucleic acids to support substrates is provided, for example, in US Pat. Nos. 5837832, 6087112, 5215882, 5707807, 5807522, 5958342. It can be found in No. 5994076, No. 6004755, No. 6048695, No. 6060240, No. 6090556 and No. 6040138.

In some embodiments, the expressed transcripts (eg, transcripts of the microRNA genes described herein) are represented in nucleic acid arrays. In such embodiments, a set of binding sites can include probes with different nucleic acids that are complementary to different sequence segments of the expressed transcript. Examples of such nucleic acids include those having a length of 15 to 200 bases, 20 to 100 bases, 25 to 50 bases, and 40 to 60 bases. Each probe sequence can also contain one or more linker sequences in addition to the sequences that are complementary to its target sequence. A linker sequence is a sequence between a sequence that is complementary to its target sequence and the surface of the support. For example, the nucleic acid array of the invention can have one probe that is specific for each target microRNA gene. However, if desired, nucleic acid arrays are at least 2, 5, 10 that are specific for any expressed transcript (eg, transcripts of the microRNA genes described herein). It can contain, 100, 200, 300, 400, 500 or more probes.

Kits In another aspect, the invention provides kits that embody methods, compositions and systems for analyzing expression of polypeptides and microRNAs as described herein. Such kits may contain the nucleic acids described herein with any or all of the following: assay reagents, buffers, probes and / or primers, and sterile. Physiological saline or another pharmaceutically acceptable emulsion base and suspension base. In addition, the kit may include explanatory material containing instructions (eg, protocol) for performing the methods described herein. For example, the kit may be a kit for amplification, detection, identification or quantification of a target mRNA or target microRNA sequence. To achieve such objectives, the kit may contain suitable primers (eg, hairpin primers), forward primers, reverse primers and probes.

In one example, the kit of the invention comprises one or more microarray slides (or alternative microarray formats) in which a plurality of different nucleic acids (each corresponding to one of the genes described above) are located. The kit can also include multiple labeled probes. Alternatively, the kit is a selection of multiple polynucleotide sequences that are suitable as probes, and labels that are suitable for optimizing the polynucleotide sequences contained or other polynucleotide sequences at the discretion of the practitioner. Can be included. In general, at least one inclusion polynucleotide sequence corresponds to a control sequence, such as a normalized gene. Exemplary labels include, but are not limited to, fluorophore, dyes, radioactive labels, enzyme tags, which are linked to nucleic acid primers.

In one embodiment, a kit suitable for amplifying the nucleic acid corresponding to the expressed RNA sample is provided. Such kits include reagents and primers suitable for use in any of the amplification methods described above. Alternatively, or in addition, the kit is suitable for amplifying the signal corresponding to hybridization between the probe and the target nucleic acid sample (eg, the target nucleic acid sample placed on a microarray).

In addition, one or more materials and / or reagents required to prepare a biological sample for gene expression analysis are optionally included in the kit. In addition, what is optionally included in these kits is to provide the required reaction mixture for amplification.
One or more enzymes suitable for amplifying nucleic acids, including various polymerases (RT, Taq, etc.), one or more deoxynucleotides, and buffers.

Typically, the kit is used to analyze gene expression patterns using mRNA or microRNA as a starting template. RNA templates may be given as either total cellular RNA or isolated RNA; both types of samples yield comparable results. In other embodiments, the methods and kits described in the present invention allow quantification of other products of gene expression, including tRNA, rRNA or other transcripts.

Optionally, the kits of the invention further include software to facilitate the generation, analysis and / or storage of data and to facilitate access to the database. The software includes logical instructions, instruction sets or suitable computer programs that can be used in the collection, storage and / or analysis of data. Comparative analysis and relationship analysis of the data is possible using the software provided.

The kit optionally contains a separate container for each individual reagent component and / or enzyme component. It would be suitable for each ingredient to be generally subdivided in its respective container. The container of the kit may optionally include at least one vial, ampoule or test tube. Flasks, bottles and other container mechanisms in which reagents can be placed and / or subdivided are also possible. The individual containers of the kit are preferably sealed and maintained for commercial sale. Suitable larger containers may include injection-molded or blow-molded plastic containers that hold the desired vials. Instructions, such as documented instructions or videotape demonstrations detailing the use of the kit of the invention, are optionally provided with the kit.

In a further aspect, the invention is the use of any composition or kit herein, or the practice of any method or assay herein, and / or any of those herein. Provided is the use of any device or kit for performing the assay or method of.

"Test sample" or "biological sample" as used herein may mean a sample of biological tissue or fluid containing nucleic acid. Such samples include, but are not limited to, tissues or body fluids isolated from animals. Biological samples also include tissue sections (eg, biopsy and death samples, etc.), frozen sections taken for histological purposes, blood, plasma, serum, sputum, stool, tears, mucus. May include urine, exudate, sheep's water, ascites, hair and skin. Biological samples also include explants and primary cell cultures and / or transformed cell cultures derived from patient tissue. Biological samples may be provided by removing a sample of cells from an animal, but biological samples are also provided by previously isolated cells (eg, by another person, at another time, and). / Or can be achieved by using cells isolated for another purpose) or by performing the methods described herein in vivo. Archived samples, such as those with treatment history or outcome history, may also be used.

The term "body fluid" or the term "body fluid" refers to a fluid obtained from the animal body, whatever the fluid. Examples of body fluids include, but are not limited to, plasma, serum, blood, lymph, cerebrospinal fluid, synovial fluid, urine, saliva, mucus, phlegm and sputum. Body fluid samples may be collected by any method of choice. Body fluid samples may be used immediately or may be stored for subsequent use. Any suitable storage method known in the art may be used to store body fluid samples: for example, the sample may be frozen at about -20 ° C to about -70 ° C. be. A suitable body fluid is extracellular fluid. In a "cell-free" fluid, a fluid in which cells are absent or the determined miRNA level is present at a low concentration that reflects that level in the fluid portion of the sample rather than the cellular portion. Includes samples. Such cell-free body fluids are generally obtained by treating cell-containing body fluids, for example by centrifugation or filtration, to remove cells. Typically, cell-free fluid does not contain any intact cells, however, some cell-free fluid may contain cell fragments or debris. Examples of extracellular fluids include plasma or serum, or body fluids from which cells have been removed.

The term "gene", as used herein, is a transcriptional regulatory sequence and / or a translational regulatory sequence and / or a coding region or untranslated sequence (eg, an intron, 5'untranslated sequence and 3'untranslated sequence. ) Containing a natural gene (eg, a genomic gene) or a synthetic gene. The coding region of a gene may be an amino acid sequence or a nucleotide sequence encoding a functional RNA (eg, tRNA, rRNA, catalytic RNA, siRNA, miRNA or antisense RNA). The gene may also be an mRNA or cDNA corresponding to a coding region (eg, exon and miRNA) that optionally comprises a 5'untranslated or 3'untranslated sequence linked to the coding region. The gene may also be an amplified nucleic acid molecule produced in vitro containing all or part of the coding region and / or a 5'untranslated or 3'untranslated sequence linked to the coding region. The term also includes pseudogenes that are dysfunctional analogs of known genes that have lost their protein-encoding ability or are otherwise no longer expressed in cells.

"Expression profile", as used herein, refers to a genomic expression profile, eg, a microRNA expression profile. Profiles may be obtained by any convenient means for determining the level of nucleic acid sequences, such as quantitative hybridization of microRNAs, cRNAs, quantitative PCR, ELISA for quantification, and the like. , Profiles may allow analysis of differential gene expression between two samples. A sample of subject or patient, eg, a cell or population thereof, eg, tissue, is assayed. Samples are collected by any method that is convenient as is known in the art. The nucleic acid sequence of interest is a nucleic acid sequence that is found to be predictive, including the nucleic acid sequences of those described herein, in which case the expression profile includes all of the listed nucleic acid sequences. Including, it may contain expression data for 5, 10, 20, 25, 50, 100 or more of the listed nucleic acid sequences. The term "expression profile" may also mean measuring the abundance of a nucleic acid sequence in a measured sample.

"Differential expression" refers to a qualitative or quantitative difference in the temporal gene expression pattern and / or the cell's gene expression pattern within and between cells and tissues. Thus, a differentially expressed gene may qualitatively have altered expression, including activation or inactivation, in normal tissue relative to diseased tissue, for example. Expression of a gene may be turned on or off in a particular state relative to another state, thus allowing comparison of two or more states. Qualitatively regulated genes will exhibit patterns of expression within conditions or cell types that may be detectable by standard techniques. Several genes will be expressed in one state or one cell type, not in both one state and one cell type. In the alternative, the difference in expression is, for example, regulated or upregulated, resulting in an increased amount of transcript or downregulated, resulting in a reduced amount of transcript. In that respect, it can be quantitative. The degree of difference in expression only needs to be large enough to be quantified by standard characterization techniques, such as by expression arrays, quantitative reverse transcriptase PCR, Northern analysis and RNase protection.

"Nucleic acid" or "oligonucleotide" or "polynucleotide", as used herein, refers to at least two nucleotides that are covalently linked together. By indicating a single strand, the sequence of complementary strands is also defined. Therefore, the nucleic acid also includes the indicated single-stranded complementary strand. Many variants of nucleic acid may be used for the same purpose as a given nucleic acid. Thus, nucleic acids also include substantially identical nucleic acids and their complements. Single chains provide probes that may hybridize to target sequences under stringent hybridization conditions. Thus, nucleic acids also include probes that hybridize under stringent hybridization conditions.

Nucleic acids may be single-stranded or double-stranded, or may contain moieties consisting of both double-stranded and single-stranded sequences. Nucleic acid is DNA (both genomic DNA and cDNA), RNA, or nucleic acid is a combination of deoxyribonucleotides and ribonucleotides, as well as uracil, adenine, thymine, cytosine, guanine, inosin, xanthin, hypoxanthin, isositocin and isoguanine. It may be a hybrid that may contain various combinations of bases, including. Nucleic acid may be obtained by chemical synthesis or recombination.

The term "primer" refers to a nucleic acid that provides a free 3'hydroxy end that can hybridize to a complementary nucleic acid molecule at its 3'end and can be extended by a nucleic acid polymerase. show. As used herein, the amplification primer may anneal to the 5'or 3'region of the gene (plus and minus chains, respectively, or vice versa, minus and plus chains, respectively). It is a pair of nucleic acid molecules that can be formed and contain a short region in between. Under the right conditions, and with the right reagents, such primers allow amplification of nucleic acid molecules with nucleotide sequences sandwiched by these primers. For in situ methods, cell or tissue samples can be prepared, immobilized on a support (eg, glass slide, etc.) and then contacted with a probe capable of hybridization to RNA. Alternative methods for amplifying the nucleic acid corresponding to the expressed RNA sample include, for example, the method described in US Pat. No. 7,897,750.

The term "probe" as used herein, as used herein, is usually via one or more types of chemical bonds, usually through complementary base pairing. The oligonucleotides that can bind to the target nucleic acid of the complementary sequence through the formation of hydrogen bonds are shown. The probe may bind to a target sequence that lacks complete complementarity with the probe sequence, depending on the stringency of the hybridization conditions. There may be many base pair mismatches that would prevent hybridization between the target sequence and the single-stranded nucleic acids described herein. However, if the number of mutations is so high that hybridization cannot occur even under the hybridization conditions of the smallest stringency, then the sequence is not a complementary target sequence. The probe may be single-stranded, or it may be partially single-stranded and partially double-stranded. The strandedness of the probe is determined by the structure, composition and nature of the target sequence. The probe may be directly labeled, for example, with biotin, which the streptavidin complex may later bind to, or
May be indirectly labeled.

When "complementary" or "complementary" is described herein to refer to a nucleic acid, Watson-Crick base pairing (eg, AT /) between nucleotides or nucleotide analogs of a nucleic acid molecule. It may mean U and CG) or Hoogsteen base pairing. Perfect complement or fully complementary may mean 100% complementary base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

As described herein, a "stringent hybridization condition" means that the first nucleic acid sequence (eg, a probe) is a second nucleic acid sequence (eg, a target), eg, in a complex mixture of nucleic acids. ) Shows the conditions for hybridization. Stringent conditions are sequence-dependent, vary from situation to situation, and can be suitably selected by those of skill in the art. Stringent conditions may be selected to be about 5 ° C to 10 ° C lower than the thermal melting point (Tm) for a particular sequence at a defined ionic strength pH. Tm may be the temperature (under defined ionic strength, pH and nuclear concentration) at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium. Is in excess, so at Tm, 50% of the probe is used in equilibrium). Stringent conditions are sodium ions less than about 1.0 M at pH 7.0-8.3, eg, sodium ion concentrations (or other salts) of about 0.01 M-1.0 M. And at least about 30 ° C. for short probes (eg, about 10 to 50 nucleotides) and at least about 60 ° C. for long probes (eg, greater than about 50 nucleotides). There may be. Stringent conditions may also be achieved by the addition of destabilizing agents (eg, formamide, etc.). For selective or specific hybridization, the positive signal may be at least 2-fold to 10-fold background hybridization. Exemplary stringent hybridization conditions include: 50% formamide when incubating at 42 ° C, 5 × SSC and 1% SDS, or 65 ° C when incubating. Is accompanied by washing at 65 ° C. with 5 × SSC, 1% SDS, which is 0.2 × SSC and 0.1% SDS. However, some factors other than temperature, such as salt concentration, can affect the stringency of hybridization, and those skilled in the art will appreciate a variety of factors to achieve similar stringency. You can choose.

As used herein, the term "reference value" refers to a value that statistically correlates to a particular outcome when compared to assay results. In a preferred embodiment, reference values are determined from statistical analysis of studies comparing expression of microRNAs with known clinical outcomes. The reference value may be a threshold score value or a cutoff score value. Typically, the reference value will be a threshold where one outcome is more certain when it is exceeded (or below), and an alternative threshold is more certain when it is below.

In one embodiment, the reference level is at one or more circulating miRNA levels expressed as the average of the levels of circulating miRNAs obtained from a sample taken from a control population of healthy (disease-free) subjects. There may be. In another embodiment, the reference level is determined at different time points, eg, the level in the same subject before the assay, eg, the level required before the subject develops the disease, or before starting treatment. It may be a level or the like. In general, samples are normalized by common factors. For example, cell-free body fluid samples are normalized by body fluid volume, and cell-containing samples are normalized by protein content or cell number. Nucleic acid samples may also be normalized to internal control nucleic acids.

As disclosed herein, differences in the level of one or more polypeptides or RNAs (mRNAs or microRNAs) imply a disease or stage thereof. The expression "difference in level" indicates a difference in the amount of a particular marker (eg, nucleic acid, etc.) in a sample, as compared to a control level or reference level. For example, the amount of a particular biomarker may be present in an elevated or decreased amount in a sample of a patient with a neoplastic disease compared to a reference level. In one embodiment, the "level difference" is at least about 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%. , 60%, 75%, 80% 100%, 150%, 200% or more, with certain biomarkers present in the sample as compared to the control (eg, reference value). It may be a difference. In one embodiment, the "level difference" may be a statistically significant difference between the amounts of biomarkers present in the sample as compared to the control. For example, the measured levels of biomarkers are from about 1.0 standard deviation, about 1.5 standard deviation, about 2.0 standard deviation, or about 2 of the mean of either control or reference group. .5 If it deviates from the standard deviation, the difference may be a statistically significant difference. For miRNA measurements, levels may be measured from real-time PCR as Ct values, in which case the Ct values may be normalized to ΔCt values as described in the examples below.

Drug Screening The present invention provides methods for identifying compounds that are useful for treating melanoma or for inhibiting endothelial recruitment, cell infiltration or metastatic angiogenesis.

Candidate compounds for screening (eg, proteins, peptides, peptide mimetics, peptoids, antibodies, small molecules or other drugs), any of a number of approaches to combinatorial library methods known in the art. Can be obtained by using in. Such libraries include the following libraries: peptide libraries, peptoid libraries, novel non-peptide skeletons that have peptide functionality but are resistant to enzymatic degradation. Molecules library), spatially addressable parallel solid-phase or liquid-phase libraries, synthetic libraries obtained by deconvolution or affinity chromatography selection, and "one-beads-one-compound" libraries. .. For example, Zuckermann et al., 1994, J. Mol. Med. Chem. , 37: 2678-2685, and Lam, 1997, Antineoplastic Drug Des. , 12: 145. Examples of various methods for synthesizing molecular libraries are, for example, DeWitt et al., 1993, PNAS USA, 90: 6909; Erb et al., 1994, PNAS USA, 91: 11422; Zuckermann et al., 1994, J. Mol. Med. Chem. , 37: 2678; Cho et al., 1993, Science, 261: 1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl. , 33: 2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. , 33: 2061; and Gallop et al., 1994, J. Mol. Med. Chem. , 37: 1233. A library of compounds may be presented in solution (eg, Houghten, 1992, Biotechniques, 13: 412-421), or beads (Lam, 1991, Nature, 354: 82-84), chips (Foodor, 1993, Nature, 364: 555-556), bacteria (US Pat. No. 5,223,409), spores (US Pat. No. 5,223,409), plasmids (Cull et al., 1992, PNAS USA, 89: 1865). -1869) or phage (Scott and Smith, 1990, Science, 249: 386-390; Devlin, 1990, Science, 249: 404-406; Cwilla et al., 1990, PNAS US
A, 87: 6378-6382; Felici, 1991, J. Mol. Mol. Biol. , 222: 301-310; and may be presented in US Pat. No. 5,223,409).

To identify useful compounds, test cells expressing the test compound express a reporter gene encoded by a nucleic acid operably linked to the promoter of a marker gene selected from the above-mentioned metastasis-promoting or metastasis-suppressing factors. It can be contacted with the containing system. This system can be an in vitro cell line model or an in vivo animal model. The cell can express the gene in its natural state or can be modified to express a recombinant nucleic acid. The recombinant nucleic acid can contain a nucleic acid encoding a reporter polypeptide for a heterologous promoter. The expression level of the miRNA, polypeptide or reporter polypeptide is then measured.

For polypeptides, the expression level can be determined at either the mRNA level or the protein level. Various methods for measuring mRNA levels in cells, tissue samples or body fluids are widely known in the art. To measure mRNA levels, cells can be lysed and the levels of mRNA in the lysate, or the levels of mRNA in RNA purified or semi-purified from the lysate, eg, hybridization assay ( It can be determined by detectively labeled gene-specific DNA or RNA probes) and quantitative or semi-quantitative RT-PCR (using appropriate gene-specific primers). Alternatively, quantitative or semi-quantitative in situ hybridization assays are performed using tissue sections or non-dissolved cell suspensions and detectable (eg, fluorescent or enzyme) labeled DNA or RNA probes. Can be done. Further mRNA quantification methods include RNA protection assay (RPA) and SAGE. Methods of measuring protein levels in cell or tissue samples are also known in the art.

Control levels, the levels obtained in the manner described above, to treat melanoma or to determine the efficacy of candidate compounds for inhibiting endothelial recruitment, cell infiltration or metastatic angioplasty. For example, it can be compared with the level obtained in the absence of the candidate compound). The compound is considered to be effective if (i) the level of the metastasis inhibitor is lower than the control or reference value, or (ii) the level of the metastasis promoter is higher than the control or reference value. Be identified. The efficacy of the compounds thus identified can be further confirmed using an in vitro cell culture model or an in vivo animal model as disclosed in the Examples below.

Example 1 Materials and Methods This example describes the materials and methods used in Examples 2 to 11 below.

Compound

Animal Studies All mouse experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Rockefeller University. 6-8 week old age-matched and sex-matched mice were used for primary tumor growth and metastasis assays as previously described (Minn et al., 2005; Tavazoie et al., 2008). .. See extended experimental procedure.

Cell Culture All cancer cell lines were cultured as previously described (Tavazoie et al., 2008). 293T cells and human umbilical vein endothelial cells (HUVEC) were maintained under standard conditions. MiRNA studies and gene knockdown / overexpression studies in cell lines and in vitro functional assays are detailed in the extended experimental procedure.

Microarray Hybridization To identify miRNAs that are deregulated across various highly metastatic derivatives, small RNAs were concentrated from total RNA from MeWo and A375 cell lines and subjected to profiling by LC Sciences. To identify potential genetic targets for miR-199a-3p, miR-199a-5p and miR-1908, total RNA obtained from the MeWo cell line was labeled by the Rockefeller University Genomics Score Facility and Illumina HT-12. It was subjected to hybridization to a v3 Expression BeadChip array. See Extended Experimental Procedures for thresholds and criteria used to reach miRNA targets and mRNA targets.

Analysis of miRNA expression in human melanoma skin lesions All human clinical samples used in this study were obtained, processed and analyzed according to IRB guidelines. Total RNA was extracted from paraffin-embedded cross-sections of primary melanoma skin lesions previously resected from the patient at MSKCC and specific miRNA expression levels were measured using TaqMan miRNA Analysis (Applied Biosystems). Was analyzed in a blinded manner. Kaplan-Meier curves representing metastasis-free survival data for each patient as a function of miRNA expression in primary tumors were generated using the GraphPad Prism software package.

In vivo LNA Treatment 4 × 10 After injecting ⁴ MeWo-LM2 cells into the tail vein, NOD-SCID mice are delivered in 0.1 mL PBS at a combined dose of 12.5 mg / kg, miR.
It was treated by intravenous administration twice a week for 4 weeks with in vivo optimized LNA (Exiqon) which is antisense against -199a-3p, miR-199a-5p and miR-1908.

Histology H & E stained 5 μm thick lung tissue sections for overall macroscopic metastatic nodule visualization. For in vivo endothelial content analysis, lung sections were subjected to antibodies to MECA-32 (Developmental Studies Hybridoma Bank, The University of Iowa, IA) (which labels mouse endothelial cells) and antibodies to human vimentin. Double staining with (Vector Laboratories), which labels human melanoma cells. See extended experimental procedure.

Data analysis All data are expressed as mean ± SEM. The Kolmogorov-Smirnov test was used to determine the significance of the difference in the cumulative distribution of metastatic vessel density. The prognostic potential of miRNAs that predict the outcome of metastases was tested for significance using the Mantel-Cox Logrank test. The one-sided Man-Whitney t-test was used to determine significance for non-Gaussian bioluminescence measurements. A one-sided Student's t-test was used for all other comparisons. P-values less than 0.05 were considered statistically significant.

In vivo selection, experimental metastasis and primary tumor growth assays All mouse experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Rockefeller University. In vivo selection was performed as previously described to generate a large number of metastatic derivatives from two independent human melanoma cell lines (Minn et al., 2005, Nature, 436, 518-524; Pollack and Fiddler). , 1982, J. Natl. Cancer Inst., 69, 137-141). Briefly, 1 × 10 ⁶ pigmented MeWo melanoma parent cells or non-pigmented A375 melanoma parent cells were resuspended in 0.1 mL PBS and immunocompromised NOD-SCID mice aged 6 to 8 weeks. Was injected intravenously. After the lung metastases were formed, the nodules were isolated and the cells were grown in vitro, resulting in a first generation lung metastatic derivative (LM1). The LM1 cells were then subjected to another in vivo selection by injecting 2 × 10 ⁵ cells into NOD-SCID mice via the tail vein, thereby producing metastatic nodules and subsequent isolation. Obtained a second-generation lung metastatic derivative (LM2). For the A375 cell line, a third in vivo selection was performed to obtain a highly metastatic A375-LM3 derivative.

To monitor translocation in vivo by bioluminescence imaging, A375 parent cells and MeWo parent cells and their transposable derivatives were transduced with a retroviral construct expressing a luciferase reporter (Ponomarev et al., 2004, Eur). J Nucl Med Mol Imaging, 31, 740-751). For all metastatic experiments, lung or systemic colonization was monitored and quantified over time by non-invasive bioluminescence imaging as previously described (Minn et al., 2005). To determine if in vivo selection was achieved, 4 × 10 ⁴ MeWo parent cells or MeWo-LM2 cells and 1 × 10 ⁵ A375 parent cells or A375-LM3 cells in 0.1 mL PBS. It was resuspended and injected via the lateral tail vein into 6-8 week old NOD-SCID mice. 4 × 10 ⁴ MeWo parents overexpressing miR-199a, miR-1908, miR-214 or control hairpins for an experimental metastasis assay to examine the effect of the presumed promoter miRNA on lung colonization. Cells, miR-199a-3p, miR-199a-5p, miR-19
08 or 4x10 ⁴ MeWo-LM2 cells in which expression of the control sequence is silenced, and 2x10 ⁵ inhibited for miR-199a-3p, miR-199a-5p, miR-1908 or control sequence A375-LM3 cells were resuspended in 0.1 mL PBS and injected into the tail vein into 6-8 week old NOD-SCID mice. For epistasis experiments, 1 × 10 ⁵ MeWo-LM2 cells expressing shRNA targeting ApoE, DNAJA4 or control sequences, or expressing siRNAs that inhibit LRP1 or control sequences in the context of miRNA inhibition. They were intravenously injected into 6-8 week old NOD-LSID mice. For ApoE pretreatment experiments, MeWo-LM2 cells were incubated at 37 ° C. in the presence of ApoE or BSA at 100 μg / mL. Twenty-four hours later, 4 × 10 ⁴ cells were injected into 7-week-old NOD-SCID mice via the tail vein. To clarify the effect of pretreatment of highly metastatic melanoma cells with LNAs targeting miR-199a-3p, miR-199a-5p and miR-1908, on metastasis, MeWo-LM2 cells. Each LNA was individually transfected with an LNA cocktail targeting all three miRNAs or with a control LNA. After 48 hours, 1 × 10 ⁵ cells (resuspended in 0.1 mL PBS) were intravenously administered to 7-week-old NOD-SCID mice for study of metastatic colonization in the lung. Alternatively, it was administered by intracardiac injection to 7-week-old athymic nude mice for a systemic metastasis assay. To clarify the effect of genetic deletion of ApoE on metastasis, 5 × 10 ⁴ B16F10 mouse melanoma cells were added to 8-week-old C57BL / 6-WT mice or C57BL / 6-ApoE − / − mice. It was injected intravenously. For the study of primary tumor growth, 1 × 10 ⁶ parental MeWo cells overexpressing miR-199a, miR-1908 or control hairpins were mixed 1: 1 with Matrigel and 6 week old immunodeficiency NOD. -Subcutaneous injection into the lower right flank of SCID mice. Animals were palpated weekly for tumorigenesis, and after tumorigenesis, fairly large tumors were measured twice a week. Tumor volume was calculated as (minimum diameter) ² x (maximum diameter) / 2.

MiRNA inhibition and gene knockdown by lentivirus 293T cells were seeded on 10 cm plates to achieve 60% confluency. Prior to transfection, the cell medium was replaced with fresh antibiotic-free DMEM medium supplemented with 10% FBS. 6 μg of Vector A, 12 μg of Vector K, and 12 μg of suitable miR-Zip (System Biosciences, Mountain View, CA) or shRNA plasmid construct (MSKCC).
HTS Core Facility, New York, NY) in 60 μL of TransIT-293 transfection reagent (MIR 2700, Minis Bio).
LLC, Madison, WI) was used for co-transfection. The cells were incubated at 37 ° C. for 48 hours and the virus was collected by centrifuging the cell medium at 2000 g for 10 minutes and then filtering the virus through a 0.45 μm filter. 1 × 10 ⁵ cancer cells were transduced with 2 mL of appropriate virus for 6 hours in the presence of 10 μg / mL polybrene (TR-1003-G, Millipore, Billerica, MA). After 48 hours, 2 μg / mL puromycin (P8833, Sigma-Aldrich, St Louis, MO) was added to the cell medium for lentivirus selection. The cells were kept in puromycin-selected state for 72 hours. The following miR-Zip sequence was used:
miR-Zip- 199a-3p: 5'-GATCCGACAGTAGCCTGCACATTAGTCACTTCCTGTCAGTAACCAATGTGCAGACTACTGTTTTTTGAATT-3'

miR-Zip- 199a-5p: 5'-GATCCGCCCAGTGCTCAGACTACCCGTGCCTTCCTGTCAGGAACAGGTAGTCTGAACACTGGGTTTTTGAATT-3'

miR-Zip-1908 5'-GATCCGCGGCGGGAACGGCGATCGGCCCTTCCTGTCAGGACCAATCGCCGTCC CCGCCGTTTT
TGAATT-3'

The following shRNA sequence was used:

shAPOE ¹ :
5'CCGGGAAGGAGTTGAAGGCCTACAACTCGAGTTGTAGGCCTTCAACTCCTTCTTTTT3'

shAPOE ² :
5'CCGGGCAGACACTGTCTGAGCAGGTCTCGAGACCTGCTCAGACAGTGTCTGCTTTTT3'

shDNAJA4 ¹ :
5'CCGGGCGAGAAGTTTAAACTCATATCTCGAGATATGAGTTTAAACTTCTCGCTTTTT3'

shDNAJA4 ² :
5'CCGGCCTCGACAGAAAGTGAGGATTCTCGAGAATCCTCACTTTCTGTCGAGGT TTTT3'

Overexpression of miRNA and genes by retrovirus 6 μg vector VSVG, 12 μg vector Gag-Pol, and 12 μg pBabe plasmid containing the coding sequence for human ApoE, DNAJA4, or an empty vector, or miR-199a, MiR-Vec containing the precursor sequence of miR-214, miR-1908 or control hairpin was co-transfected into 60% confluent 293T cells using 60 μL of TransIT-293 transfection reagent. The cells were incubated at 37 ° C. for 48 hours, then the virus was collected and transduced into cancer cells for 6 hours in the presence of 10 μg / mL polybrene. After 48 hours, 2 μg / mL puromycin or 10 μg / mL blastsidedin (15205, Sigma-Aldrich, St Louis, MO) was added to the cell medium for retrovirus selection. The cells were kept in puromycin-selected state for 72 hours or in blastsaidin-selected state for 7 days. The following cloning primers were used for overexpression of the coding sequences of ApoE and DNAJA4:
ApoE CDS Fwd: 5'-TCATGAGGATCCATGAAGGTTCTGTGGGCT-3'
ApoE CDS Rev: 5'-T AGC AG AATTCTC AGTGATTGTCGCTGGG-3'
DNAJA4 CDS Fwd: 5'-ATCCCTGGATCCATGTGGGAAAGCCTGACCC-3'
DNAJA4 CDS Rev: 5'-TACCATGTCGACTCATGCCGTCTGGCACTGC-3'

LNA-based miRNA knockdown Mature miR-199a-3p, mature miR-199a-5p, mature miR-1908 or LNA complementary to control sequences (426917-00, 426918-00, 426878-00, respectively) And 1990050; Exiqon, Vedbaek, Denmark) to 50% confluent MeWo-LM2 cancer cells cultured in antibiotic-free medium with Lipofectamine ™ 2000 Transfection Reagent (11668-09, Invitrogen, Carlsbad, CA). Was transfected with a final concentration of 50 nM. After 8 hours, the transfection medium was replaced with fresh medium. After 48 hours, 1 × 10 ⁵ cells were intravenously infused into NOD-SCID mice to assess metastatic colonization in the lung, or cardiac to athymic nude mice to assess systemic metastasis. It was injected by internal injection. Cells were used 96 hours after transfection for in vitro assays for cell infiltration and endothelial recruitment.

mRNA knockdown based on siRNA siRNA targeting LRP1, LRP8, VLDLR, LDLR or control sequences was transfected into cancer cells or HUVECs at a final concentration of 100 nM using Lipofectamine ™ 2000 Transfection Reagent. After 5 hours, the transfection medium was replaced with fresh medium. Cells were subjected to Matrigel infiltration assay and endothelial mobilization assay 96 hours after transfection. Cells transduced with siRNA targeting LRP1 or control sequences in the context of miRNA inhibition were injected into the tail vein for a colonization assay in the lung 72 hours post-transfection. Control non-targeted siRNAs were obtained from Dharmacon. The following LRP1 and LRP8 target sequences were used:
siLRP1 ¹ : 5'-CGAGGACGAUGACUGCUUA-3';
siLRP1 ² : 5'-GCUAUGAGUUUAAGAAGUU-3';
S1LRP8 ¹ : 5'-CGAGGACGAUGACUGCUUA-3';
siLRP8 ² : 5'-GAACUAUUCACGCCUCAUC-3'.
Cell proliferation assay To clarify the effect of miR-199a or miR-1908 overexpression and combinatorial LNA-induced miRNA inhibition on cell proliferation, 2.5 × 10 ⁴ cells were seeded in triplets in 6-well plates. The live cells were counted after 5 days. Incubate 3 × 10 ⁴ melanoma MeWo-LM2 cells or endothelial cells in the presence of ApoE (100 μM) or BSA (100 μM) to assess the effect of recombinant ApoE addition on melanoma cell proliferation or endothelial cell proliferation. did. Live cells were counted after 8 hours, 24 hours, 48 hours, 72 hours and 120 hours.

Matrigel infiltration assay Cancer cells were subjected to serum starvation for 12 hours in DMEM-based medium with 0.2% FBS. Transwell infiltration chambers (354480, BD Biosciences, Bedford, MA) were pre-equilibrated prior to assay initiation by adding 0.5 mL of starvation medium to the top and bottom chambers. After 30 minutes, the medium in the upper chamber was removed, 0.5 mL of medium containing 1 × 10 ⁵ cancer cells was added into each Matrigel-coated transwell insert and incubated at 37 ° C. for 24 hours. For neutralizing antibody and / or recombinant protein experiments, antibody / recombinant protein was added to each well at the start of the assay at the concentrations shown below: 5 μg / mL-40 μg / mL. Anti-ApoE 1D7 (Heart Assay, Universality of Ottawa), 5 μg / mL-40 μg / mL anti-IgG (AB-108-C, R & D Systems, Minneapolis, MN), 100 μM recombinant human ApoE3 (46) View, CA) and 100 μM BSA (A2153, Sigma-Aldrich). When the assay was complete, Matrigel-coated inserts were washed with PBS, cells on the top surface of each insert were scraped, and the inserts were fixed in 4% paraformaldehyde for 15 minutes. The insert was then cut out and secured to the slide using a VectorSheld fixing medium containing DAPI (H-1000, Vector Laboratories, Burlingame, CA). The base surface of each insert was imaged using an inverted fluorescence microscope (Zeiss Axiovert 40 CFL) at a magnification of 5x, whereby three representative images were taken for each insert. The number of infiltrated cells was quantified using ImageJ (NIH).

Endothelial Mobilization Assay 5 × 10 ⁴ cancer cells were seeded in 24-well plates approximately 24 hours prior to the start of the assay. HUVEC was grown to 80% confluency and subjected to 16 hours of serum starvation in EGM-2 medium supplemented with 0.2% FBS. The HUVEC was then pulsed with Cell Tracker Red CMTPX dye (C34552, Invitrogen) for 45 minutes. In the meantime, the cancer cells were washed with PBS, 0.5 mL of EGM-2 medium with 0.2% FBS was added to each well, and 3.0 μm HTS.
Fluoroblock inserts (351151, BD Falcon, San Jose, CA) were placed in each well. 1 × 10 ⁵ HUVECs (resuspended in 0.5 mL starvation medium) were seeded into each transwell insert and the mobilization assay was run at 37 ° C. for 16-18 hours. For neutralizing antibody and / or recombinant protein experiments, the antibody / protein was then added to each well at the appropriate concentrations below as shown in the figure: 40 μg / mL anti-ApoE 1D7, 40 μg. / ML anti-IgG, 100 μΜ rhApoE3 and 100 μΜ BSA. When the assay was completed, the inserts were treated and analyzed as described for the Matrigel infiltration assay above (see Matrigel Infiltration Assay).

Endothelial Migration Assay HUVECs subjected to serum starvation were pulsed with Cell Tracker Red CMTPX dye for 45 minutes and HTS Fluroblock transwells at concentrations of 1 × 10 ⁵ HUVECs in 0.5 mL starvation medium for each insert. It was sown in the insert. The assay was run at 37 ° C. for 16-18 hours and the inserts were treated and analyzed as described above (see Matrigel Infiltration Assay).

The chemotaxis assay HUVEC was subjected to 16 hours of serum starvation in EGM-2 medium with 0.2% FBS and labeled with Cell Tracker Red CMTPX dye for 45 minutes. In the meantime, the indicated amount (1 μg-5 μg) of recombinant human ApoE3 or BSA was mixed with 250 μL Matrigel (356231, BD Biosciences) and allowed to solidify at the bottom of a 24-well plate for 30 minutes. Then 250 μL of HUVEC EGM-2 medium containing 0.2% FBS was added to each Matrigel coated well and a 3.0 μM HTS Fluroblock insert was fitted into each well. 1 × 10 ⁵ HUVECs (resuspended in 0.5 mL starvation medium) were seeded into each insert and allowed to migrate along the Matrigel gradient at 37 ° C. for 16-18 hours. When the assay was complete, inserts were secured to slides as described above and analyzed (see Matrigel Infiltration Assay).

Endothelial Adhesion Assay HUVEC was seeded on 6-well plates to form a monolayer. Cancer cells were subjected to serum starvation for 30 minutes in DMEM-based medium with 0.2% FBS and Cell Tracker.
Pulsed for 45 minutes with a Green CMFDA dye (C7025, Invitrogen). 2 × 10 ⁵ cancer cells (resuspended in 0.5 mL starvation medium) were seeded on each endothelial monolayer. Cancer cells were adhered to the HUVEC monolayer at 37 ° C. for 30 minutes. Then, the endothelium monolayer was gently washed with PBS and fixed with 4% paraformaldehyde for 15 minutes. Each well was then covered with PBS and eight images were taken for each endothelial monolayer using an inverted fluorescence microscope (Zeiss Axiovert 40 CFL) at a magnification of 10x. The number of cancer cells adhering to HUVEC was quantified using ImageJ.

Anoyquis assay 1 × 10 ⁶ MeWo cells overexpressing miR-199a, miR-1908 or control hairpins were seeded on a low adhesive plate containing a cell medium supplemented with 0.2% methylcellulose. .. After placing in suspension for 48 hours, the number of dead and live cells was counted using trypan blue.

Serum Starvation Assay To clarify the effect of miR-199a and miR-1908 on the serum starvation ability of melanoma cells, 1 × 10 ⁵ MeWo parent cells overexpressing miR-199a, miR-1908 or control hairpins were used. The cells were seeded in quadruples on 6-well plates and incubated in a starved DMEM-based medium with 0.2% FBS for 48 hours, after which the number of viable cells was counted using tripan blue. To clarify the effect of the addition of recombinant ApoE3 on the survival of melanoma cells or endothelial cells in serum starvation conditions, 3 × 10 ⁴ MeWo-LM2 cells or endothelial cells were subjected to low serum conditions (0.2% FBS). ) Incubated in the presence of ApoE3 (100 μM) or BSA (100 μM). The number of live cells was counted after 8 hours, 16 hours and 24 hours.

Colony forming assay 50 MeWo parent cells overexpressing miR-199a, miR-1908 or control hairpins were seeded in quadruple 6 cm plates. After 2 weeks, cells were washed with PBS, fixed with 6% glutaraldehyde and stained with 0.5% crystal violet. The number of positively stained colonies was counted.

miRNA microarray hybridization To identify miRNAs that exhibit deregulated expression across various highly metastatic melanoma cell line derivatives, a number of independent metastatic derivatives and their respective parent MeWo cell populations and parent A375 cells. Total RNA from the population was used to concentrate small RNAs, which were then labeled and hybridized to a custom microarray platform of microfluids by LC sciences. The array was designed to detect 894 mature miRNAs corresponding to the miRNA transcripts listed in Sanger miRBase Release 13.0. Of all the probes analyzed, the probes corresponding to 169 miRNAs produced signals above the background threshold across the large number of cell lines analyzed. Raw signal intensities, which correspond to probe hybridization, were median-normalized for each cell line. An upregulation threshold of more than twice the median-normalized expression value was used to identify miRNAs commonly induced in multiple metastatic derivatives for two independent human melanoma cell lines.

Microarray-based Gene Target Prediction for miR-199a and miR-1908 Total RNA, respectively, to identify potential genes targeted by miR-199a-3p, miR-199a-5p and miR-1908, respectively. Illumina HT-12 extracted from MeWo cell lines with loss or gain of function of miRNA
It was sent to the Genomics Score Facility at Rockefeller University for hybridization to the v3 Expression BeadChip microarray. Raw signal intensities, which correspond to probe hybridization, were then median-normalized for each cell line sample. Three sets of microarray profiles were compared: (1) MeWo control cells against MeWo cells overexpressing miR-199a or miR-1908, (2) miR-199a-3p, miR-199a-5p or miR-1908. MeWo-LM2 control cells for MeWo-LM2 cells expressing short hairpins (miR-Zip) to be targeted, and (3) MeWo parent cells for MeWo-LM2 cells. Based on the median-normalized expression values obtained from these arrays, the following criteria were used to reach possible target genes common to miR-199a and miR-1908: (1) miR. Genes that are down-regulated by more than 1.5-fold difference to each individual overexpression of -199a and miR-1908, (2) inhibition of both miR-199a-3p and miR-1908 or miR-199a Genes that are upregulated by more than 1.5-fold difference in either inhibition of -5p and miR-1908, and (3) physiologically higher of the above three miRNAs relative to MeWo parent cells. A gene that is down-regulated by a difference of more than 1.5 times in LM2 cells expressing the level.

Analysis of miRNA and mRNA expression in cell lines Total RNA was extracted from various cell lines using the miRvana kit (AM1560, Applied Biosystems, Austin, TX). Expression levels of mature miRNAs were quantified using the Taqman miRNA expression assay (4429775-0002228, Applied Biosystems). RNU44 was used as an endogenous control for normalization. For mRNA expression analysis, 600 ng of total RNA was reverse transcribed using the cDNA First-Strand Synthesis Kit (18080-051, Invitrogen) and approximately 200 ng of the resulting cDNA was then SYBR Green PCR Master Mix (4309155). , Applied Biosystems) and appropriate primers. Each reaction was performed in quadruples and mRNA expression was quantified by performing real-time PCR amplification using the ABI Prism 7900HT Real-Time PCR System (Applied Biosystems). GAPDH was used as an endogenous control for normalization. The following primers were used:
ApoE Fwd: 5'-TGGGTCGCTTTTGGGATTAC-3'
ApoE Rev: 5'-TTC AACTCCTTC ATGGTCTCG-3'
DNAJA4_Fwd: 5'-CCAGCTTCTCTTCACCCATG-3'
DNAJA4 Rev: 5'-GCCAATTTCTTCGTGACTCC-3'
GAPDH Fwd: 5'-AGCCACATCGCTCAGACAC-3'
GAPDH Rev: 5'-GCCCAATACGACCAAATCC-3'
LRPl Fwd: 5'-TTTAACAGC ACCGAGTACCAG-3'
LRPl Rev: 5'C AGGC AGATGTC AGAGC AG-3'
LRP8 Fwd: 5'-GCTACCCTGGCTACGAGATG-3'
LRP8 Rev: 5'-GATT AGGG ATGGGCTCTTGC-3'
ELISA
The conditioned cancer cell medium was prepared by incubating the cells in serum starved DMEM-based medium with 0.2% FBS for 24 hours. The level of ApoE in the conditioned medium is determined by the APOE ELISA kit (IRAPKT031, Innovative).
Research, Novi, Michigan).

Luciferase Reporter Assay A heterologous luciferase reporter assay was performed as previously described (Tavazoie et al., 2008). Briefly, full-length 3'UTRs and CDSs of ApoE and DNAJA4 were cloned into the psiCheck2 dual luciferase reporter vector (C8021, Promega, Madison, WI) downstream of the sea shiitake mushroom cipherase reporter. 5 × 10 ⁴ parental MeWo cells, MeWo-LM2 cells, miR-199a, miR-1908 or MeWo cells overexpressing control hairpins, and miR-199a-3p, miR-199a-5p, miR-1908 or MeWo-LM2 cells expressing miR-Zip hairpins targeting the control sequence were transfected with 100 ng of each specific reporter construct using the TransiT-293 transfection reagent. Twenty-four hours after transfection, cells were lysed and the ratio of sea shiitake mushroom luciferase expression to firefly luciferase expression was determined using the dual luciferase assay (E1910, Promega). The presumed miRNA binding site in each target construct is a complementary miRNA seed sequence (miR-199a-3p: 5'-CAGUAGUC-3'; miR-199a-5p: 5'-CCAGUGUU-3'; miR- 1908: 5'-GGCGGGGA-3') identified by alignment. The miRNA complementary sites in each target construct were mutated using the QuickChange Multi Site-Directed Mutagenesis Kit (200514, Agilent Technologies, Santa Clara, CA). Based on miRNA seed sequence complementarity analysis, the CDS of ApoE was mutated at position 141 (from CTG to ACT) and the 3'UTR of ApoE was mutated at position 83 (from GCC to ATA) and at position 98 (from CTG). Mutate the CDS of DNAJA4 at position 373 (from CGC to TAT) and mutate at position 917 (from CTG to AGA) and mutate the 3'UTR of DNAJA4 at position 576 (from CTG to ACA). Mutations were made at position 1096 (from CTG to TCT), at position 1396 (from CGC to TGT), and at position 1596 (from CTG to TGT). The following primers were used to clone the 3'UTR and CDS of ApoE and DNAJA4:
ApoE CDS Fwd: 5'-AGTACCTCGAGGGGATCCTTGAGTCCTACTC-3'
APOE CDS Rev: 5'-TAATTGCGGCCGCTCAGACAGTGTCTGCACCCAG-3'
DNAJA4_CDS_Fwd: 5'-TAATATCTCGAGATGTGGGAAAGCCTGACCC-3'
DNAJA4 CDS Rev: 5'-CAATTGCGGCCGCTCATGCCGTCTGGCACTGC-3'
APOE 3'UTR Fwd: 5'-TTAGCCTCGAGACGCCGAAGCCTGCAGCCA-3'
APOE 3'UTR Rev: 5'-TTACTGCGGCCGCTGCGTGAAACTTGGTGAATCTT-3'
DNAJA4 3'UTR Fwd: 5'-TAATATCTCGAGCGTGGTGCGGGGCAGCGT-3'
DNAJA4 3'UTR Rev: 5'-CAATTGCGGCCGCTTATCTCTCATACCAGCTCAAT-3'
The following primers were used to mutate the miRNA binding site in each target:
APOE CDS mut: 5'-
GCCAGCGCTGGGAACTGGCAACTGGTCGCTTTTGGGATTACCT-3'
APOE 3'UTR mutl: 5'-
CAGCGGGAGACCCTGTCCCCATACCAGCCGTCCTCCTGGGGTG-3'
APOE 3'UTR_mut2: 5'-
TCCCCGCCCCAGCCGTCCTCACAGGGTGGACCCTAGTTTAATA-3'
DNAJA4_CDS_mutl: 5'-
GGGATCGGTGGAGAAGTGCCTATTGTGCAAGGGGCGGGGGATG-3'
DNAJA4_CDS_mut2: 5'-
GTAGGGGGCGGGGAACGTGTTATCCGTGAAGAGGTGGCTAGGG-3'
DNAJA4 3'UTR mutl: 5'-
CAGGGCCAACTTAGTTCCTAACATTCTGTGCCCTTCAGTGGAT-3'
DNAJA4 3'UTR_mut2: 5'-
ACAGTTTGTATGGACTACTATCTTAAATTATAGCTTGTTTGGA-3'
DNAJA4 3'UTR_mut3: 5'-
TAATTATTGCTAAAGAACTATGTTTTAGTTGGTAATGGTGTAA-3'
DNAJA4 3'UTR_mut4: 5'-
CAGCTGCACGGACCAGGTTCCATAAAAACATTGCCAGCTAGTGAG-3'

Analysis of miRNA expression in human melanoma skin lesions All human clinical samples used in this study were obtained, processed and analyzed according to institutional IRB guidelines. Paraffin-embedded cross-sections of primary melanoma skin lesions from 71 human patients were obtained from MSKCC. These samples were deparaffinized by 5 consecutive xylene washes (5 minutes each). After deparaffinization, malignant tumor-containing areas were identified by H & E staining, dissected, and total RNA was extracted from the anatomy using the RecoverAll Total Nucleic Acid Isolation Kit (AM1975, Applied Biosystems). Expression levels of mature miR-199a-3p, miR-199a-5p and miR-1908 in each sample were quantified in a blinded manner using the Taqman miRNA assay. R
NU44 was used as an endogenous control for normalization. The expression levels of each miRNA were compared between primary melanomas that tended to metastasize and those that did not. The Kaplan-Meier curve was plotted as a function of expression levels for each miRNA in each patient's tumor using patient metastasis-free survival data. Metastatic recurrences to sites such as lung, brain, bone and soft tissue have been previously recorded, and such metastatic recurrences are retrospective of the relationship between identified miRNA expression levels and metastatic recurrences. Enables targeted analysis.

Histological animals were perfused with PBS and then fixed with 4% paraformaldehyde injected by intracardiac injection followed by intratracheal injection. Lungs were incised, incubated overnight at 4 ° C. in 4% paraformaldehyde, embedded in paraffin and sliced into 5 μm thick serial sections. Lung sections were H & E stained for overall macroscopic metastatic nodule visualization. For endothelial content analysis in metastatic nodules formed in mice by human melanoma MeWo cells, representative lung sections were presented with primary antibodies against MECA-32 (Developmental Studies Hybridoma Bank, The Universality of Iowa, IA). Labels mouse endothelial cells) and double-stained with a primary antibody against human vimentin (VP-V684, Vector Laboratories), which labels human melanoma cells. Various Alexa Fluor dye-conjugated secondary antibodies were used to detect the primary antibody. Fluorescence was measured using a Zeiss laser scanning confocal microscope (LSM510) to determine vascular density within the metastatic nodules and the MECA-32 signal within each metastatic nodule (thus having a human contour). (Shown based on co-staining with vimentin) was quantified in a blinded manner using ImageJ (NIH). Representative lung sections were stained with MECA-32 for endothelial content analysis in metastatic nodules formed by B16F10 mouse melanoma cells in wild-type mice and ApoE genetic null mice, and MECA within each nodule. The -32 signal, which borders on the pigmentation of the cells, was quantified in a blinded manner. The total vascular area (which is given as the percentage area covered by the blood vessels to the total area of each metastatic nodule), background removal (the radius of a 1-pixel rolling ball), and given as a cutoff. Obtained by the use of thresholds. Metastatic nodules were defined as regions with a total area of more than 2000 μm ² in any region. For large nodules, at least four representative images were obtained and their average vessel density was calculated.

Matrigel plug assay in vivo 10 μg / mL recombinant human ApoE3 (4696, BioVision), 10 μg / mL BSA (A2153, Sigma Aldrich) or 400 ng / ml VEGF, as shown by Matrigel (356231, BD Biosciences). Was mixed with. 400 μL of Matrigel containing the indicated recombinant protein was subcutaneously injected just above the ventral flank of immunocompromised NOD-SCID mice. The plug was removed 3 days after injection and fixed in 4% paraformaldehyde for 48 hours. The plug was then embedded in paraffin and made into continuous sections with a thickness of 5 μm. Cross-section sections of the plugs were immunohistochemically stained with a primary antibody against the mouse endothelial antigen MECA-32 (Developmental Studies Hybridoma Bank, The University of Iowa, IA) and detected by a peroxidase-conjugated secondary antibody. , Subsequently visualized by DAB oxidation. To quantify the degree of endocellular infiltration into each Matrigel plug, the number of endothelial cells was counted in 4-5 random fields for each plug and endothelial cells per given plug area. The average number of was calculated.

Tissue culture SK-Mel-334 primary human melanoma strains were established from soft tissue metastases of Braf mutant melanoma in one patient at MSKCC. After minimal expansion culture in vitro, cells were selected in vivo to give rise to the lung metastatic derivative SK-Mel-334.2 (Pollac and Fiddler, 1982). The SK-Mel-239 vemurafenib-resistant clone (C1) is a transfer from Paulikos Poulikakos (Mount Sinai Medical School) and is a transfer of B-Raf ^{V600E / +} ; Pten ^{− / −} ; CDKN2A ^{− / −} The cell line was transferred by Marcus Rosenberg (Yale University). All other cell lines used were purchased from ATCC.

ApoE's Elisa
Extracellular ApoE levels in serum-free conditioned medium obtained from melanoma cells treated with DMSO, GW3965 or T0901317 (1 μM each) are quantified 72 hours after treatment using an ApoE ELISA kit (Innovative Research). did.

Lung and brain tissue samples from Western blotting mice were homogenized on ice in RIPA buffer (Sigma-Aldrich) supplemented with a protease inhibitor (Roche). Mouse adipose tissue was homogenized on ice with TNET buffer (1.5 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% Triton, protease inhibitor). Total protein lysate (2 μg) was separated by SDS-PAGE, transcribed on PVDF membrane and blotting with anti-mouse ApoE antibody (ab20874, Abcam) and anti-tubulin α / β antibody (2148, Cell Signaling).

Analysis of ApoE expression in melanoma clinical samples All clinical samples were obtained, processed and analyzed in strict accordance with IRB guidelines. Primary melanoma skin lesions were pre-excised from the patient at MSKCC, formalin-fixed, paraffin-embedded, and sectioned into 5 μm thick slide pieces. ApoE protein expression was evaluated by double-blind immunohistochemical analysis using D6E10 anti-ApoE antibody (ab1906, Abcam).

Tissue Chemistry Animals were perfused intracardiac with PBS followed by perfusion with 4% paraformaldehyde (PFA). The fixed lungs were embedded in paraffin and made into continuous sections with a thickness of 5 μm. Macroscopic lung metastatic nodules were visualized by H & E staining. To analyze endothelial cell content in tumors, tumor growth and apoptosis, paraffin-embedded sections of primary tumors were subjected to antibodies to MECA-32 (Developmental Studies Hybridoma Bank, Universality of Iowa), antibodies to KI-67. (Ab15580, Abcam) and antibody against truncated caspase-3 (9661, Cell Signaling), respectively.

Tail vein transfer assay The melanoma cells used for the in vivo transfer assay were transduced with a stably expressed retroviral construct encoding the luciferase reporter gene (Ponomarev et al., 2004), thereby the present inventors. In vivo progression of melanoma cells could be monitored by bioluciferase imaging. The following number of melanoma cells (resuspended in 100 μL PBS) were injected intravenously via the tail vein: 4 × 1
0 ⁴ MeWo cells, 2.5 × 10 ⁵ HT-144 cells, 2 × 10 ⁵ SK-Mel-334.2 cells, 5 × 10 ⁴ B16F10 cells, and 1 × 10 ⁵ Individual YUMM cells. MeWo cells, HT-144 cells and SK-Mel-334.2 cells were injected into 6-8 week old sex-matched NOD scid mice, while B16F10 cells and YUMM cells were 6-8 weeks old. It was injected into sex-matched C57BL / 6 mice. In all experiments assessing the effect of GW3965 on metastasis formation, mice were pretreated with a control diet or GW3965 supplement diet (20 mg / kg) for 10 days. To assess the effect of GW3965 treatment on brain metastases, 1 × 10 ⁵ MeWo brain metastatic derivatives were intracardiac injected into thymic nude mice. Immediately after infusion, mice were randomly assigned to control diet or GW3965 supplement diet (100 mg / kg). To determine if oral delivery of GW3965 could inhibit the progression of early metastases, NOD Scid mice were intravenously injected with 4 × 10 ⁴ MeWo cells and the cells colonized the lungs over 42 days. The mice were then blindly assigned to control diet treatment or GW3965 supplementation diet treatment (100 mg / kg).

Orthotopic metastasis assay To clarify the effect of GW3965 treatment on lung colonization by melanoma cells isolated from orthotopic sites, 1 × 10 ⁶ MeWo cells expressing luciferase reporter were used in NOD Scid. Subcutaneous injection was performed into the lower flanks on both sides of the mouse. When a tumor of approximately 300 mm ³ in volume was formed, the tumor was resected and mice were randomly assigned to control diet or GW3965 supplemental diet (100 mg / kg). One month after tumor resection, the lungs were removed and colonization in the lungs was measured by Exvivo-bioluminescence imaging. To histologically confirm the degree of colonization in the lungs of melanoma, the lungs were then fixed overnight with 4% PFA, paraffin-embedded, 5 μM serial sections and stained for human vimentin. (VP-V684, Vector Laboratories).

Preparation of Dacarbazine-Resistant Melanoma Cells Dacarbazine-resistant B16F10 mouse melanoma cells were prepared by continuously culturing the cells in the presence of DTIC (D2390, Sigma-Aldrich, St. Louis, MO). Initially, cells were treated with 500 μg / mL DTIC for 1 week. After this initial DTIC treatment, the remaining (about 10%) live cells were restored for 1 week, after which 750 μg / mL DTIC was added to the cell medium for 5 days. Following this high dose treatment, cells were restored in the presence of a low dose of DTIC (100 μg / mL) for 1 week. The cells were then continuously cultured in cell medium containing 200 μg / mL DTIC for at least 1 month prior to transplanting the cells into mice. DTIC was added to fresh cancer cell medium every 3 days. For tumor growth experiments, 5 × 10 ⁴ B16F10 parent cells and DTIC-resistant cells were subcutaneously injected into the lower flank of 7-week-old C57BL / 6 mice. After the formation of small tumors measuring 5 mm ³ to 10 mm ³ in volume, mice were randomly assigned to the following treatment groups: (1) control diet + vehicle (ip); (2) control diet + DTIC. (Ip) (50 mg / kg); (3) GW3965 supplemental bait (100 mg / kg) + vehicle (ip). DTIC was dissolved in water in the presence of citric acid (1: 1 by weight) and administered daily by intraperitoneal injection. DTIC-resistant MeWo human melanoma cell line clones were obtained after DTIC treatment in mice with MeWo tumors measuring 600 mm ³ to 800 mm ³ in volume. After the initial tumor shrinkage in response to daily DTIC dosing (50 mg / kg, ip) during the first two weeks, the tumor eventually developed resistance and resumed growth. At that point, tumor cells were isolated and a DTIC resistant MeWo cell line was established. Cells were expanded in vitro for 1 week in the presence of DTIC (200 μg / mL) and then 5 × 10 ⁵ DTIC resistant MeWo cells were reinjected into 8-week-old Nod SCID gamma mice. Tumor volume is 5 mm ^3-10 mm ³
Mice were blindly assigned to the following treatment groups: (1) control diet + (2) control diet + DTIC (50 mg / kg); (3) GW3965 supplement diet (100 mg / kg). To elucidate the effect of DTIC on tumor growth by parental non-selected MeWo cells, 5 × 10 ⁵ MeWo cells were subcutaneously injected into Nod SCID gamma mice and the mice were 5 mm ^3-10 mm ³ in volume. After a tumor was formed, it was treated with a control vehicle or DTIC (50 g / kg). DTIC was administered daily as described above in a cycle consisting of 5 consecutive daily treatments with a 2-day washout interval. Tumor growth was measured twice a week.

Gene initiation model of melanoma progression Tyr :: CreER; B-Raf ^{V600E / +} ; Pten ^{lox / +} / Tyr :: CreER; B-Raf ^{V600E / +} ; Pten ^{lox / lox} Conditional model of melanoma progression is Dankort et al. (2009) ) Was previously established and characterized. Briefly, melanoma in these mice was injected intraperitoneally at 25 mg / kg dosed with 4-HT (H6278, 70% isomers, Sigma-Aldrich, St Louis, MO) in peanut oil for 3 consecutive days. It was induced at 6 weeks of age by injection. A stock solution of 4-HT was prepared by heating 4-HT at 45 ° C. for 5 minutes and dissolving at 50 mg / mL in 100% EtOH by mixing. Once dissolved, this stock 4-HT solution was diluted 10-fold in peanut oil, resulting in a 5 mg / mL 4-HT working solution to be subsequently injected into mice. After the first 4-HT injection, mice were blindly assigned to receive either a control diet or a diet supplemented with GW3965 (100 mg / kg). Mice were examined three times a week for the presence and progression of melanoma lesions. On day 35, dorsal skin samples were collected from control treated and GW3965 treated mice, fixed in 4% PFA and photographed at 10X. Percentages of pigmented melanoma lesion area from total skin area were quantified using ImageJ. For survival analysis, mice were monitored daily for melanoma progression and euthanized according to standard physical condition scores, taking into account the initial signs of dying and discomfort associated with progression of melanoma loading. After death, lungs, brain and salivary glands were collected and examined for the presence of macroscopic melanoma lesions.

Mouse Genotyping All mouse genotyping was performed using standard PCR conditions as recommended by Jackson Labs. The following genotyping primers were used for each PCR reaction:
Tyr :: CreER; B-Raf ^{V600E / +} ; Pten ^{lox / +} mouse and Tyr :: CreER; B-Raf ^{V600E / +} ; Pten ^{lox / lox} mouse:
B-Raf Forward: 5'-TGA GTA TTT TTG TGG CAA CTG C-3'
5-a / Reverse: 5'-CTC TGC TGG GAA AGC GGC-3'
Pten Forward: 5'-CAA GCA CTC TGC GAA CTG AG-3'
Pten Reverse: 5'-AAG TTT TTG AAG GCA AGA TGC-3'
Cre Transgene Forward: 5'-GCG GTC TGG CAG TAA AAA CTA TC-3'
Cre Transgene Reverse: 5'-GTG AAA CAG CAT TGC TGT CAC TT-3'
Internal Positive Control Forward: 5'-CTA GGC CAC AGA ATT GAA AGA TCT-3'
Internal Positive Control Reverse: 5'-GTA GGT GGA AATTCT AGC ATC ATC C-3'
ApoE-/-Mouse:
Common Forward: 5'-GCC TAG CCG AGG GAG AGC CG-3'
Wild-type Reverse: 5'-TGT GAC TTG GGA GCT CTG CAG C-3'
Mutant Reverse: 5'-GCC GCC CCG ACT GCA TCT-3'
LXRα-/-mouse:
Common Forward: 5'-TCA GTG GAG GGA AGG AAA TG-3'
Wild-type Reverse: 5'-TTC CTG CCC TGG ACA CTT AC-3'
Mutant Reverse: 5'-TTG TGC CCA GTC ATA GCC GAA T-3'
LXRβ-/-mouse:
Common Forward: 5'-CCT TTT CTC CCT GAC ACC G-3'
Wild-type Reverse: 5'-GCA TCC ATC TGG CAG GTT C-3'
Mutant Reverse: 5'-AGG TGA GAT GAC AGG AGA TC-3'

Cell proliferation and viability assays To demonstrate the effects of GW3965, T0901317 and bexarotene on in vitro cell growth, 2.5 × 10 ⁴ melanoma cells were seeded in triplets in 6-well plates, DMSO, GW3965, The cells were cultured in the presence of T0901317 or bexarotene at 1 μM each. After 5 days, the number of live and dead cells was counted using tripan blue (72-57-1, Sigma-Aldrich), which selectively labels dead cells.

Cell Infiltration Assays Cell infiltration assays were performed using the Transwell Matrigel Infiltration Chamber System (354480, BD Biosciences) as described in detail previously (Pencheva et al., 2012). Briefly, various melanoma cells were cultured in the presence of DMSO, GW3965, T0901317 or bexarotene in 1 μΜ for 56 hours, after which the melanoma cells were cultured in starvation medium (0. Switched to 2% FBS). After starvation, cells were seeded into Matrigel-coated transwell inserts and the infiltration assay proceeded at 37 ° C. for 24 hours. 40 μg / mL 1D7 anti-ApoE blocking antibody (Heart Institute, Universality of Ottawa, Ottawa, Canada) or 40 μg / mL anti-IgG control antibody (AB-108-C, R & D Systems, Min) for ApoE antibody neutralizing experiments. , MN) were added to each transwell insert at the start of the assay.

Endothelial Mobilization Assays Endothelial mobilization assays were performed as previously described (Pencheva et al., 2012; Png et al., 2012). Melanoma cells were treated with DMSO, GW3965, T0901317 or bexarotene at 1 μΜ for 56 hours, after which 5 × 10 ⁴ cells were seeded in 24-well plates in the presence of each drug and 16 prior to starting the assay. Adhered over time. HUVEC cells were subjected to overnight serum starvation in EGM-2 medium containing 0.2% FBS. The next day, 1 × 10 ⁵ HUVEC cells were placed in a 3.0 μm HTS Fluroblock transwell migration insert (351151, BD Falcon, San Jose, CA) fitted into each well containing cancer cells at the bottom. Sown. HUVEC cells were allowed to migrate towards cancer cells at 37 ° C. for 20 hours, after which the inserts were treated as previously described (Pencheva et al., 2012). 40 μg / mL 1D7 anti-ApoE blocking antibody (Heart Institute, Universality of Ottawa, Ottawa, Canada) or 40 μg / mL anti-IgG control antibody (AB-108-C, R & D Systems, Min) for ApoE antibody neutralizing experiments. , MN) were added to each transwell insert at the start of the assay.

Gene knockdown shRNA based on lentivirus shRNA, as previously described (Pencheva et al., 2012; Png et al., 2012), 6 μg Vector A, 12 μg Vector K and 12 μg shRNA plasmid in the HEK-293T package. Incorporated into lentiviral particles prepared by transfecting shRNA cells. Transduction of shRNA by lentivirus was performed for 6 hours in the presence of 10 μg / mL polybrene (TR-1003-G, Millipore, Billerica, MA) as previously described (Pencheva et al., 2012). The cells were expanded and cultured for 72 hours after transduction, and lentivirus selection was performed by culturing the cells in the presence of 2 μg / mL puromycin (P8833, Sigma-Aldrich) for 72 hours.

The following shRNA sequence was used:
Human:
Sh ₁ LXR α: 5'-
CCGGCCGACTGATGTTCCCACGGATCTCGAGATCCGTGGGAACATCAGTCGGTT TTT-3'
sh ₂ LXR α: 5'-
CCGGGCAACTCAATGATGCCGAGTTCTCGAGAACTCGGCATCATTGAGTTGCTT TTT-3'
sh ₃ LXR β: 5'-
CCGGAGAGTGTATCACCTTCTTGAACTCGAGTTCAAGAAGGTGATACACTCTTT TTT-3'
sh ₂ LXR β: 5'-
CCGGGAAGGCATCCACTATCGAGATCTCGAGATCTCGATAGTGGATGCCTTCTT TTT-3'
shApoE: 5'-
CCGGGCAGACACTGTCTGAGCAGGTCTCGAGACCTGCTCAGACAGTGTCTGCTT TTT-3'
mouse:
sh_mLXRα: 5'-
CCGGGCAACTCAATGATGCTGAGTTCTCGAGAACTCAGCATCATTGAGTTGCTT TTT-3'
sh_mLXRβ: 5'-
CCGGTGAGATCATGTTGCTAGAAACCTCGAGGTTTCTAGCAACATGATCTCATTTTTG-3'
sh_mApoE: 5'-
CCGGGAGGACACTATGACGGAAGTACTCGAGTACTTCCGTCATAGTGTCCTCTT TTT-3'

Gene expression analysis by qRT-PCR:
RNA was extracted from whole cell lysate using the Total RNA Purification Kit (17200, Norden, Thorold, Canada). Then, 600 ng of total RNA was reverse transcribed into cDNA using the cDNA First-Strand Synthesis Kit (18080-051, Invitrogen) and quantitative real-time PCR amplification was performed by ABI Prism 7900HT Real-Time PCR System (Applied Biosystems). , TX) as previously described (Pencheva et al., 2012). Each PCR reaction was performed in quadruple. Gene expression was normalized to GAPDH used as an endogenous control.

The following primers were used:
Human:
ApoE Forward: 5'-TGGGTCGCTTTTGGGATTAC-3'
ApoE Reverse: 5'-TTCAACTCCTTCATGGTCTCG-3'
GAPDH Forward: 5'-AGCCACATCGCTCAGACAC-3'
GAPDH Reverse: 5'-GCCCAATACGACCAAATCC-3'
LXRα Fwd: 5'-GTTATAACCGGGAAGACTTTGC-3'
LXRα Rev: 5'- AAACTCGGC ATC ATTGAGTTG-3'
LXRβ_Fwd: 5'-TTTGAGGTATTTGAGTAGCGG-3'
LXRβRev: 5'-CTCTCGCGGAGTGAACTAC-3'
mouse:
ApoE Forward: 5'-GACCCTGGAGGCTAAGGACT-3'
ApoE Reverse: 5'-AGAGCCTTCATCTTCGCAAT-3'
GAPDH Forward: 5'-GCAC AGTC AAGGCCGAGAAT-3'
GAPDH Reverse: 5'-GCCTTCTCC ATGGTGGTGAA-3'
LXRα Forward: 5'-GCGCTCAGCTCTTGTCACT-3'
LXRα Reverse: 5'-CTCCAGCCACAAGGACATCT-3'
LXRβ Forward: 5'-GCTCTGCCTAC ATCGTGGTC-3'
LXRβ Reverse: 5'-CTCATGGCCCAGCATCTT-3'
ABCA1 Forward: 5'-ATGGAGCAGGGAAGACCAC-3'
ABCA1 Reverse: 5'-GTAGGCCGTGCCAGAAGTT-3'

ApoE promoter activity assay ApoE promoter (which consists of sequences extending to 980 base pairs upstream and 93 base pairs downstream of the ApoE gene) is restricted to pGL3-Basic vectors (E1751, Promega Corporation, Madison, WI). It was cloned upstream of firefly luciferase using an enzyme and a SacI restriction enzyme. The multi-enhancer element 1 (ME.1) and multi-enhancer element 2 (ME.2) were then cloned using MluI and SacI restriction enzymes just upstream of the ApoE promoter. ApoE promoter drive by LXR agonist and ME. 1 / MW. To assess 2-driven transcriptional activation, 5 × 10 ⁴ MeWo cells were seeded in 24-well plates. The next day, 100 ng of pGL3-ME. 1 / ME. The 2-ApoE promoter construct and 2 ng of pRL-CMV sea shiitake luciferase construct (E2661, Promega) were co-transfected into cells in quadruple under each condition in the presence of DMSO, GW3965 or T0901317 at 1 μΜ. To assess transcriptional activation by LXRα or LXRβ, 5 × 10 ⁴ MeWo cells expressing control shRNA or ThenRNA targeting LXRα or LXRβ were seeded in 24-well plates. The next day, 200 ng of pGL3-ME. 1 / ME. The 2-ApoE promoter construct and 2 ng of pRL-CMV sea shiitake luciferase construct were co-transfected into cells in quadruplicate with each condition in the presence of DMSO, GW3965 or T0901317 at 1 μΜ. After 24 hours, cells were lysed and cell lysates were analyzed for firefly luciferase activity and sea luciferase activity using Dual Luciferase Assay System (E1960, Promega) and Bio-Tek Synergy NEO Microplate Reader. The signal of firefly luciferase was normalized to the signal of sea shiitake mushrooms. All data are expressed for the luciferase activity ratio measured in DMSO treated control cells.

The following cloning primers were used:
ApoE-promoterForward: 5'-TCA TAG CTA GCG CAG AGC CAG GAT TCA CGC CCT G-3'
ApoE-promoter Reverse: 5'-TGG TCC TCG AGG AAC CTT CATCTT CCT GCC TGT GA-3'
ME.1 Forward: 5'-TAG TTA CGC GTA GTA GCC CCC ATC TTT GCC-3'
ME.1 Reverse: 5'-AAT CAG CTA GCC CCT CAG CTG CAA AGC TC-3'
ME.2 Forward: 5'-TAG TTA CGC GTA GTA GCC CCC TCT TTGCC-3'
ME.2 Reverse: 5'-AAT CAG CTA GCC CTT CAG CTG CAA AGCTCT G-3'

Tumor histochemistry Tumors were resected from mice and fixed in 4% paraformaldehyde at 4 ° C. for 48 hours. The tumor was then embedded in paraffin and made into continuous sections with a thickness of 5 μm. For endothelial cell content analysis in tumors, tumor sections were stained with a primary antibody against MECA-32, a mouse endothelial cell marker (Developmental Studies Hybridoma Bank, The University of Iowa, IA) and counterstained by DAPI nuclear staining. .. To reveal tumor cell proliferation and apoptosis, tumor sections were sampled with antibodies against the proliferative marker Ki-67 (Abcam, abl5580, Cambridge, MA) and antibodies against the apoptotic marker cleavage caspase-3 (9661). Staining with Cell Signaling, Antibody, MA), respectively. Various Alexa Fluoro dye-conjugated secondary antibodies were used to detect the primary antibody. Fluorescence, inverted fluorescence microscope (Zeiss)
Axiovert 40 CFL) was used for MECA-32 and Ki-67 staining at a magnification of 5x and for truncated caspase-3 staining at a magnification of 10x. Endothelial cell content and tumor growth rate were quantified by calculating the average percentage of MECA-32 positive stained area or Ki-67 positive stained area from total tumor area. Tumor apoptosis was measured by counting the number of cells expressing truncated caspase-3 per given tumor area.

Analysis of ApoE expression in primary melanoma lesions Human primary melanoma skin lesions were resected from melanoma patients at MSKCC, fixed in formalin, embedded in paraffin, and made into continuous sections with a thickness of 5 μm. To reveal ApoE protein expression, the sample was first deparaffinized by two consecutive xylene washes (5 minutes each) and then a series of ethanol washes (100%, 95%, 80% and 70%). It was rehydrated by EtOH). The ApoE antigen was recovered by incubating the sample in the presence of proteinase K (5 μg / mL) at room temperature for 20 minutes. Slides _were incubated in ₃ % H2O2 solution to inactivate endogenous peroxidase activity. The slides were then blocked three times in a row in avidin block solution, biotin block solution and horse serum block solution for 15 minutes each at room temperature (SP-2001, Vector Laboratories, Burlingame, CA). ApoE was detected by staining with D6E10 anti-ApoE antibody (ab1908, Abcam). At this time, the antibody was used overnight at 4 ° C. in PBS at a dilution of 1: 100. The primary antibody was then recognized by incubating the slides with a peroxidase-conjugated secondary antibody (PK-4002, Vector Laboratories) and manifested by a DAB (SK-4105, Vector Laboratories) oxidation reaction. Slides were imaged at a magnification of 10x and analyzed in a double-blind fashion. ApoE expression was quantified by counting the number of DAB-positive cells and measuring the area of extracellular ApoE staining. The total ApoE staining signal was expressed as a percentage staining area per given tumor area determined based on matching H & E staining slides for each sample. A Kaplan-Meier curve showing metastasis-free survival time for patients was generated by plotting recurrence-free survival data for each patient as a function of ApoE expression in the patient's primary melanoma lesion. Patients with tumors having lower ApoE levels than the population's median ApoE expression were classified as ApoE negative, whereas patients with melanoma expressing ApoE above the median were classified as ApoE positive. Based on the patient's history recorded prior to metastatic recurrence to sites such as lung, brain, bone, soft and subcutaneous tissue and skin, we have developed ApoE at the site of primary melanoma and metastatic recurrence. The relationship between them could be clarified retroactively.

Example 2 In vivo selection (Pollac and Fiddler, 1982) to identify miRNA regulators of melanoma metastasis in which endogenous mir-1908, mir-199a-3p and mir-199a-5p promote human melanoma metastasis. , Used in conjunction with the pigmented MeWo human melanoma cell line and the non-pigmented A375 human melanoma cell line to generate a number of second generation (LM2) and third generation (LM3) pulmonary metastatic derivatives. Comparison of metastatic potential of the MeWo-LM2 and A375-LM3 strains showed that these derivatives translocated significantly more efficiently than their respective parent populations in the lung colonization assay (Figure). 12A to 12B). Small RNA profiling based on hybridization of 894 mature miRNAs, followed by quantitative stem-loop PCR (qRT-PCR) to four miRNAs (miR-1908, miR-199a-3p, miR-199a- It was revealed that 5p and miR-214) were upregulated more than 2-fold for their respective parent cells in a large number of A375 and MeWo-transferred derivatives (FIG. 1A). ~ FIG. 1B, FIG. 12C). Significant induction of miR-199a-3p, miR-199a-5p, miR-214 and miR-1908 throughout a number of metastatic derivatives suggested a metastatic role for these miRNAs. Retrovirus-mediated transfection and overexpression of precursors for miR-199a-3p and miR-199a-5p (which are overexpressed simultaneously with miR-199a hairpins) and miR-1908 are bioluminance signal quantification. Caused a robust increase in metastatic colony formation in the lung based on both virus formation and overall lung histology (FIG. 1C, FIG. 12D; 9.64-fold increase for miR-1908, P = 0.016. For miR-199a, there was an 8.62-fold increase, P = 0.028), while overexpression of miR-214 had no significant effect on metastasis. Importantly, overexpression of miR-199a and miR-1908 respectively increased the number of metastatic nodules formed (Fig. 12E), consistent with their role for these miRNAs in the initiation of metastasis. rice field. These findings also revealed that miR-199a and miR-1908 were sufficient for enhanced metastatic colonization.

Various assays were then performed to determine if the endogenous levels of these miRNAs promoted metastasis. To achieve this goal, miR-1908 and each of the two miRNAs (miR-199a-3p and miR-199a-5p) resulting from the miR-199a hairpin were placed in highly metastatic cells by miR-Zip technology. Inhibited. Individual inhibition of each of these miRNAs suppressed metastatic colonization by more than 7-fold difference (FIG. 1D; P = 0.047 for miR-1908 inhibition; P = 0 for miR-199a-3p inhibition). .010; for miR-199a-5p inhibition, P = 0.015) and dramatically reduced the number of metastatic nodules formed (FIG. 12F).

Their expression was also silenced in A375 metastatic derived cell lines to determine if these miRNAs also promote metastasis in unrelated cell lines. In fact, inhibition of miR-1908, miR-199a-3p or miR-199a-5p significantly reduced the ability of metastatic A375-LM3 cells to colonize the lungs (FIG. 1E), and thus these 3 It has been demonstrated that two miRNAs are endogenous promoters of metastasis by human melanoma cells.

Given the robust functional role of miR-1908, miR-199a-3p and miR-199a-5p in facilitating melanoma metastasis in a mouse model of human cell metastasis, further assays, expression of these miRNAs in humans This was done to investigate whether it correlates with the metastatic potential of primary melanoma lesions. To achieve this goal, 71 primary melanoma skin lesions obtained from patients with Memorial Sloan-Kettering Center Center (MSKCC) were exposed to miR-1908, miR-199a-3p and miR-199a-5p. Levels were analyzed in a blinded manner by qRT-PCR. Consistent with the above functional studies, all three miRNAs were significantly induced in metastasized primary melanoma compared to non-metastatic primary melanoma (Fig. 1F; P = for miR-1908). 0.037; P = 0.0025 for miR-199a-3p; P = 0.0068 for miR-199a-5p). This suggests that upregulated expression of these miRNAs in primary lesions is an early event predicting cancer progression of melanoma.

Example 3 miR--1908, miR-199a-3p and miR-199a-5p promote cell infiltration and endothelial recruitment In this example, the assay was performed with miR--1908, miR-199a-3p and miR-199a-5p. To clarify the cellular mechanism that regulates metastasis.

First, we investigated whether these miRNAs promote metastasis by enhancing proliferation or tumor growth. On the contrary, overexpression of each miRNA reduced cell proliferation (FIG. 13A). More importantly, overexpression of miR-1998 does not increase primary tumor growth, whereas overexpression of miR-199a actually results in a significant reduction in tumor volume (35%, P <0.001). Caused (Fig. 2A). This indicates that the metastatic effects of miR-1908 and miR-199a are not secondary to tumor growth promotion or increased cell proliferation.

Next, we investigated whether these miRNAs regulate cell infiltration, an important metastatic phenotype. Metastatic LM2 cells, which express higher levels of these miRNAs, showed significantly increased ability to infiltrate Matrigel to their parent population with smaller metastases (FIG. 13B). Therefore, overexpression of miR-199a and miR-1908 individually increased the ability of parent MeWo cells to infiltrate through Matrigel (FIG. 2B; 3-fold increase for miR-199; 2 for miR-1908). Double increase). Conversely, individual inhibition of miR-199a-3p, miR-199a-5p and miR-1908 significantly reduced the infiltration capacity of MeWo-LM2 transposable melanoma cell derivatives (FIG. 2C), as well as A375. -The infiltration capacity of LM3 metastatic melanoma cell derivatives was significantly reduced (Fig. 2D).

Given the robust effects of these miRNAs on metastatic progression, further assays were performed to determine if they might regulate some further metastatic supportive phenotypes. Overexpression of miR-199a or miR-1908 results in melanoma cell adhesion to endothelial cells (FIG. 13C), resistance to anoikis (FIG. 13D), survival in serum starvation situations (FIG. 13E) or colonization (FIG. 13F). However, each miRNA dramatically increased the ability of parent MoWe cells to mobilize endothelial cells in a transwell endothelial mobilization assay (more than 3-fold increase) (FIG. 2E). Consistent with this, metastatic Mewo-LM2 cells, which physiologically overexpress miR-199a and miR-1908, are more efficient in recruiting endothelial cells to their parent cells. It was a target (Fig. 13G). Conversely, inhibition of miR-199a-3p, miR-199a-5p or miR-1908 in metastatic MeWo-LM2 cells (FIG. 2F), as well as inhibition in A375-LM3 cells (FIG. 2G), stimulates endothelial recruitment. Suppressed, this was consistent with the requirement and sufficiency of these miRNAs for increased endothelial mobilization capacity of metastatic melanoma cells.

Various assays were performed to determine whether endogenous miR-199a-3p, miR-199a-5p and miR-1908 regulate endothelial recruitment by metastatic cells in vivo to determine metastatic vascular density. Was examined by performing co-immune staining for human vimentin (which labels human MeWo melanoma cells) and mouse endothelial cell antigen (MECA-32) (which labels mouse endothelial cells). Surprisingly, inhibition of miR-199a-3p, miR-199a-5p or miR-1908 individually markedly reduced vascular density within metastatic nodules (for miR-199a-3p and miR-199a-5p). Caused 3 times on average, 4.7 times on average for miR-1908) (Fig. 2H; P <0.001 for miR-199a-3p; P <0. For miR-199a-5p. 001; P <0.001 for miR-1908), demonstrating the role of these miRNAs in promoting metastatic endothelial content and metastatic angiogenesis. Conversely, overexpression of each miRNA in low-metastatic melanoma cells dramatically increased metastatic vessel density (FIG. 13H). These findings reveal that miR-199a-3p, miR-199a-5p and miR-1908 are necessary and sufficient for increased infiltration and endothelial mobilization during melanoma progression.

Example 4 mir-1908, mir-199a-3p and mir-199a-5p target Apoe and DNAJA4 convergently and cooperatively In this example, these are systematic and unpredictable efforts. Used to identify the direct molecular target of miRNA.

miR--1908, miR-199a-3p and miR-199a-5p mediate the same set of in vitro and in vivo phenotypes, and miR-199a-3p and miR-199a-5p are the same precursor hairpins. It was hypothesized that the transfer-supporting phenotypes of these miRNAs may result from silencing of common target genes. Given that mammalian miRNAs act primarily by destabilizing target mRNA transcripts (Guo et al., 2010, Nature, 466, 835-840), transcriptome profiling of melanoma cells, respectively. It was performed in both loss-of-function and gain-of-function situations for miRNA. This results in a small set of genes that are similarly suppressed by both miR-199a and miR-1908 and also exhibit lower levels in metastatic LM2 derivatives that express higher endogenous levels of these miRNAs. Revealed (Fig. 14A). By quantitative RT-PCR, two genes, the metabolic gene apolipoprotein E (ApoE) and the heat shock protein DNAJA4, are significantly regulated by miR-199a and miR-1908 and in highly metastatic LM2 cells. It was confirmed to be dramatically silenced (FIGS. 3A and 14B-14D).

The effect of each miRNA on its presumed target stability to determine whether ApoE and DNAJA4 are directly targeted by miR-1908, miR-199a-3p and miR-199a-5p. Was examined by a heterologous luciferase reporter assay. Interestingly, overexpression of miR-199a suppresses the stability of the 3'untranslated region (UTR) and coding region (CDS) of both ApoE and DNAJA4, while overexpression of miR-1908 of ApoE. 3'UTR and 3'UTR and CDS of DNAJA4 were destabilized. Mutation of miRNA-complementary sequences in each target, consistent with direct targeting, abolished miRNA-mediated regulation (FIG. 3B). In direct testing of endogenous targeting, individual miRNA inhibition in metastatic LM2 cells resulted in increased target stability (Fig. 3C), when this increased target stability mutated the miRNA target site. It disappeared (Fig. 14E). From this, it was revealed that ApoE is directly targeted by miR-1908 and miR-199a-5p, and DNAJA4 is directly targeted by all three miRNAs (Fig. 3D). Importantly, both CDS and 3'UTRs of these genes were less stable in highly metastatic LM2 cells expressing physiologically higher levels of these three regulatory miRNAs. This indicates that the endogenous targeting of ApoE and DNAJA4 by these miRNAs is associated with melanoma metastasis (Fig. 3E).

Given the molecular convergence of miR-199a-3p, miR-199a-5p and miR-1908 to common target genes, these targets, namely ApoE and DNAJA4, have the transfer phenotypes provided by these miRNAs. Next, I investigated whether it could be mediated. Overexpression of each gene in metastatic LM2 cells caused a marked reduction in cell infiltration and endothelial recruitment phenotype (FIGS. 3F-3G, FIG. 14F). Conversely, knockdown of ApoE or DNAJA4 in hypometastatic cells using irrelevant hairpins significantly enhanced cell infiltration and endothelial recruitment (FIGS. 3H-3I, 14G). This revealed that ApoE and DNAJA4 act as endogenous suppressors of these metastasis-supporting phenotypes, consistent with their targeting by metastasis-promoting miRNAs described above.

Example 5 ApoE and DNAJA4 mediate miR-199a and miR-1908-dependent metastatic infiltration, endothelial mobilization and colonization ApoE and DNAJA4 are direct biology downstream of miR-199a and miR-1908. Various assays were performed to determine if they were target effectors, especially if these two target genes interacted with their respective miRNAs. As expected, miRNA silencing reduced the infiltration and endothelial mobilization capacity of highly metastatic melanoma cells. Importantly, knockdown of ApoE or DNAJA4 in the context of miRNA inhibition significantly impedes inhibition of infiltration (FIGS. 4A and 4C) and endothelial recruitment (FIGS. 4B and 4D) during silencing of each miRNA. rice field. Surprisingly, knockdown of either of these genes completely restored the dramatic suppression of metastatic colonization resulting from miRNA inhibition in cells depleted for miR-1908 or miR-199a-5p. (FIGS. 4E to 4F, FIG. 15E). Conversely, overexpression of ApoE or DNAJA4 causes cell infiltration and in cells that overexpress miR-1908 (FIGS. 4G-4H, 15F) or miR-199a (FIGS. 15G-15I). It was sufficient to suppress endothelial mobilization. In addition, overexpression of ApoE or DNAJA4 was sufficient to inhibit miRNA-mediated metastatic colonization (FIG. 15J). Importantly, ApoE and DNAJA4 were also required for miRNA-dependent increased cell infiltration and endothelial recruitment by highly metastatic A375-LM3 cells (FIGS. 4I-4J, 15K).

Co-immune staining of melanoma metastases (human vimentin) and endothelial cells (MECA-32) to determine whether ApoE and DNAJA4 also regulate miRNA-dependent metastatic endothelial recruitment in vivo. Performed in metastatic nodules of the lung formed in the context of miRNA inhibition by cells knocked down for each of the genes. Notably, knockdown of ApoE or DNAJA4 resulted in a significant (> 3.5-fold) increase in metastatic vessel density in metastases resulting from cells with miRNA silencing (Fig. 4K, ApoE knock). P <0.01) for both down cells and DNAJA4 knockdown cells. From these findings, ApoE and DNAJA4 are directly downstream effectors of miRNA-dependent metastatic infiltration phenotypes, colony formation phenotypes and endothelial recruitment phenotypes induced in melanoma by these metastasis-supporting miRNAs. It was revealed.

Example 6 ApoE secreted by melanoma cells is a necessary and sufficient mediator of infiltration and endothelial mobilization, while genetic deletion of ApoE promotes metastasis. ApoE is a secretory factor. As such, it was investigated whether ApoE secreted by melanoma cells could suppress infiltration and endothelial recruitment. According to it, the extracellular ApoE levels detected by ELISA are not very metastatic parent cells in metastatic LM2 cells, which express higher levels of miR-199a and miR-1908. It was 1 / 3.5 of that (Fig. 5A). Secreted ApoE levels were also significantly suppressed by endogenous miR-199a and miR-1908 (FIGS. 5B and 16A).

Next, cells by parental MeWo cells, which express high endogenous levels of ApoE (FIG. 14C), by inhibiting ApoE by using a neutralizing antibody (1D7) that recognizes the receptor binding domain of ApoE. Both infiltration (Fig. 5C, 1.68-fold increase) and endothelial recruitment (Fig. 5D, 1.84-fold increase) were increased. Conversely, the addition of recombinant human ApoE significantly suppressed infiltration and endothelial recruitment by metastatic LM2 cells, which express low endogenous ApoE levels (FIG. 14C) (FIG. 5E). Importantly, the addition of recombinant ApoE did not affect the in vitro proliferation of melanoma or endothelial cells (FIGS. 16B-16C) or survival under serum starvation conditions (FIGS. 16D-16E). This indicates that suppression of these phenotypes by recombinant ApoE is not secondary to reduced or impaired survival in proliferation. Consistent with ApoE being downstream of miR-199a and miR-1908 in epistasis, neutralization of ApoE with the ApoE neutralizing antibody 1D7 is an suppressed infiltrative phenotype and endothelium observed with inhibition of the respective miRNAs. The mobilization phenotype was significantly canceled (Fig. 5F-5G). The above findings reveal that ApoE secreted by melanoma cells is a necessary and sufficient inhibitor of miRNA-dependent infiltration and endothelial mobilization phenotypes in melanoma.

Further assays were performed to further investigate the mechanism by which DNAJA4, a poorly characterized heat shock protein, mediates endothelial recruitment and infiltration. Given the phenotypic commonality presented by ApoE and DNAJA4, it was hypothesized that DNAJA4 might play a regulatory role and increase ApoE levels. In fact, knockdown of DNAJA4 reduces both ApoE transcript levels (FIG. 16F) as well as secreted ApoE levels (FIG. 5H), while overexpression of DNAJA4 substantially reduces ApoE expression. It was raised (Fig. 16G). Consistent with DNAJA4 acting upstream of ApoE, the addition of recombinant ApoE canceled the enhanced cell infiltration and endothelial recruitment phenotypes observed with knockdown of DNAJA4 (FIGS. 5I-5J). .. Conversely, suppression of the infiltration and endothelial mobilization phenotypes observed with overexpression of DNAJA4 was significantly impeded by antibody neutralization of ApoE (FIGS. 16H-16I). These findings reveal that DNAJA4 suppresses melanoma infiltration and endothelial recruitment through positive regulation of ApoE expression and resulting secretion.

Considering the regulatory convergence of these metastasis-promoting miRNAs and DNAJA4 genes to ApoE, an assay was performed to determine whether expression of ApoE correlates with human melanoma progression. To achieve this goal, array-based published expression data for ApoE (Haqq et al., 2005, Proc. Natl. Acad. Sci. USA, 102, 6092-6097) were used for nevus lesions, primary. Analysis was performed in lesions and metastatic lesions. Consistent with its metastatic role, ApoE levels were significantly lower (P <0.025) for primary lesions and significantly lower for nevus lesions in distal organ metastases. (P <0.0003) (Fig. 5K).

Given its significant correlation with human melanoma progression, we investigated whether increasing ApoE signaling in melanoma cells could have therapeutic efficacy in suppressing melanoma metastasis. More specifically, metastatic MeWo-LM2 cells were preincubated with recombinant ApoE or BSA for 24 hours prior to infusion into mice. Surprisingly, pretreatment of cancer cells with ApoE firmly suppressed metastatic colonization by more than 300-fold (Fig. 5L). This dramatic suppression of metastasis by preincubating melanoma cells with ApoE reflects that the effect of ApoE on melanoma cells is very important for the initiation of metastasis. This is because cells pretreated with ApoE exhibit reduced infiltration capacity, which is required to initiate the metastatic event that causes colonization in the lung.

Given the robust effects of ApoE on the metastatic phenotype and the metastatic phenotype, as well as its strong association with human melanoma progression, further assays should be performed to determine the genetic deletion of systemic ApoE on melanoma progression. The effects were investigated in detail in an immuno-eligible mouse model of melanoma metastasis. Consistent with the major inhibitory role for extracellular ApoE in metastasis, circulatingly infused B16F10 mouse melanoma cells were used in ApoE genetic null mice 7 in metastatic colonization compared to their wild-type litters. It showed an increase of more than double (Fig. 5M). These findings demonstrate systemic ApoE and cancer-secreting ApoE as robust suppressors of melanoma metastasis in humans and mice.

Example 7 Extracellular ApoE Targets the LRP1 Receptor of Melanoma Cells and the LRP8 Receptor of Endothelial Cells Differently In this example, various assays are used to investigate the molecular mechanism by which ApoE suppresses translocation. Went to.

To identify the ApoE receptors that mediate infiltration, all four known ApoE receptors, namely VLDLR, LRP1, LRP8 and LDLR (Hatters et al., 2006, Trends Biochem. Sci., 31, 445-). 454; Hauser et al., 2011, Prog. Lipid Res., 50, 62-74) were knocked down in melanoma cells. Interestingly, knockdown of LRP1 did not occur at the other ApoE receptors, but abolished the cell infiltration inhibitory effect induced by recombinant ApoE (FIG. 6A). Importantly, knockdown of LRP1 in metastatic LM2 cells, which indicates low levels of ApoE, only slightly increased cell infiltration (FIG. 17A), which indicates that the effect of LRP1 is It was suggested that it is mediated by endogenous ApoE.

LRP1 was also knocked down in the context of miRNA inhibition to determine whether LRP1 mediates miRNA-dependent effects on infiltration and metastatic colonization. Knockdown of LRP1 in the context of miRNA silencing restored the suppressed infiltration phenotype resulting from miRNA inhibition (FIGS. 6B, 17B). Consistent with these in vitro results, knockdown of LRP1 markedly enhanced in vivo metastatic colonization by LM2 cells silenced for miR-1908 (FIGS. 6C, 17C). These findings reveal that LRP1 is downstream of miRNA / ApoE-dependent melanoma infiltration and metastatic colonization in epistasis.

The infiltration phenotype reflects the cell-autonomous effect of ApoE on melanoma cells, whereas the endothelial recruitment phenotype directly suggests a cell-unautonomous role of cancer-expressing ApoE on endothelial cells. Consistent with this, pretreatment of endothelial cells with ApoE significantly reduced their ability to migrate towards highly metastatic cancer cells (FIG. 6D). To identify ApoE receptors on endothelial cells that mediate the endothelial mobilization phenotype, all four known ApoE receptors were knocked down on endothelial cells. Interestingly, unlike infiltration of cancer cells, knockdown of endothelial LRP8 did not revoke any of the other receptors, but endothelial mobilization caused by miRNA silencing selectively and significantly revoked. (FIGS. 6E, 17D to 17E). These findings are consistent with the LPR8 receptor being a downstream endothelial mediator of miRNA / ApoE-dependent effects on endothelial recruitment.

Assays were then performed to determine if ApoE / LRP8 signaling may also regulate systemic endothelial migration in cancer cell-free systems. According to it, antibody neutralization of ApoE (which is present in the endothelial cell medium) significantly enhances endothelial migration (Fig. 6F), while recombinant ApoE is dependent on endothelial cell LRP8 receptors for endothelial migration. Sufficient to inhibit in the transwell assay (FIG. 6G) and gradient chemotaxis assay (FIG. 6H) in a manner. Importantly, the addition of ApoE caused a dramatic (> 40-fold difference) suppression of VEGF-induced endothelial recruitment in vivo into the subcutaneous Matrigel plug (FIG. 6I).

Given that ApoE is required and sufficient to mediate endothelial recruitment, a further assay is performed to determine if systemic ApoE may regulate metastatic angioplasty. rice field. Consistent with the robust suppression of metastatic endothelial content by ApoE secreted by melanoma cells (Fig. 4K), genetically null ApoE mice have higher vascular density compared to their wild-type litters. Shown inside those lung metastatic nodules formed by B16F10 mouse melanoma cells (FIG. 6J; 2.41 fold increase, P = 0.0055). In summary, the above findings provide a dual cell-autonomous / non-autonomous role for ApoE in the suppression of metastasis through different signaling mediated by the LRP1 receptor on melanoma cells and the LRP8 receptor on endothelial cells. Will be revealed.

Example 8 miR-199a-3p, miR-199a-5p and miR-1908 as robust prognostic and therapeutic targets in melanoma metastasis
To investigate whether the metastatic promoter miRNAs described herein can serve as clinical predictors of metastatic outcomes, the expression levels of miR-199a-3p, miR-199a-5p and miR-1908 are used. , Quantified in a blinded manner by qRT-PCR in a cohort of human melanoma samples obtained from patients at MSKCC. The relationship between these miRNA levels in primary melanoma lesions and the outcome of metastatic recurrence was then determined.

Importantly, patients with primary melanoma lesions expressing larger levels of miR-199a-3p, miR-199a-5p or miR-1908 (than the median for the population) have these primary melanomas. Patients expressing lower levels of each of the miRNAs were more likely to develop distal metastases and showed significantly shorter metastasis-free survival times (FIGS. 7A-7C, miR-199a-3p). P = 0.0032 for miR-199a-5p, P = 0.0034 for miR-199a-5p, P = 0.027 for miR-1908). Surprisingly, the total expression levels of these three miRNAs showed the strongest prognostic potential in stratifying patients at high risk for metastatic recurrence from those at very low risk (). 7D, P <0.0001). These clinical findings are consistent with the functional cooperation between these miRNAs in the regulation of cancer progression, suggesting their usefulness for these molecules as clinical prognostic biomarkers for melanoma metastasis. There is.

Given the current lack of effective treatment options to prevent melanoma metastasis, and the strong prognostic values of these three regulatory miRNAs in melanoma metastasis, these miRNAs are used with antisense LNA treatment. It was therapeutically targeted (Elmer et al., 2008 (a); Elmer et al., 2008 (b)). Highly metastatic MeWo-LM2 cells pretreated with LNA oligonucleotides that are antisense against mature miR-199a-3p, miR-199a-5p or miR-1908 are roughly 4 minutes in metastatic activity. Showed a decrease to 1. Given the clinical evidence of cooperativity between these miRNAs, the effect of silence of all three miRNAs on metastatic progression was investigated. Notably, co-transfection of LNAs to all three miRNAs suppressed metastatic colonization by more than 70-fold difference, which is why endogenous miR-199a-3p, miR-199a. Dramatic synergies and co-operation between -5p and miR-1908 were revealed (Fig. 7E, P = 0.004). Importantly, inhibition of these miRNAs by triple LNA pretreatment did not result in reduced in vitro proliferation (FIG. 18A). This indicates that this dramatic metastasis-suppressing phenotype is not secondary to impaired proliferation. Combinatorial LNA-mediated miRNA targeting in an independent A375 metastatic derivative line also significantly inhibited colonization in the lung (FIG. 18B).

Next, we investigated whether combinatorial LNA-induced miRNA inhibition could suppress systemic melanoma metastasis to multiple distal organs. In fact, by intracardiac infusion of hypermetastatic melanoma cells pretreated with a cocktail of LNAs targeting these three regulatory miRNAs, endogenous miR-199a-3p, miR-199a-5p and miR-1908. Was found to promote systemic melanoma metastasis (Fig. 7F). Combinatorial LNA-mediated inhibition of these three miRNAs caused a decrease in the number of systemic metastatic lesions at distal sites such as the brain and bone (FIGS. 7H-7I) (FIG. 7G).

A further assay was performed to investigate the therapeutic efficacy of systemically administered in vivo optimized LNAs in preventing melanoma metastasis. To achieve this goal, highly metastatic MeWo-LM2 cells were injected into mice. The next day, mice were intravenously administered at a low total dose (12.5 mg / kg) twice a week for 4 weeks by LNA targeting miR-199a-3p, miR-199a-5p and miR-1908. Treated by. Notably, combinatorial LNA treatment reduced colonization in the lung by a 9-fold difference (Fig. 7J, P = 0.031) with no apparent signs of toxicity (Fig. 18C). Taken together, the findings reveal a novel miRNA-dependent regulatory network focused on ApoE signaling to control cell-autonomous and cell-non-autonomous features of melanoma metastatic progression (Fig. 7K). The above basic studies have identified a set of miRNAs with strong prognostic and therapeutic potential in the clinical management of melanoma.

Example 9 miRNA-dependent targeting of ApoE / LRP1 signaling promotes cancer cell infiltration and endothelial recruitment through the induction of CTGF In this example, connective tissue growth factor (CTGF) is the cancer cell infiltration and endothelial. It was identified as a downstream mediator of ApoE / LRP1 signaling in recruitment. CTGF expression levels as determined by qRT-PCR analysis and ELISA are mediated by ApoE / LRP1 signaling (FIGS. 8A, 8B and 8C). In addition, ApoE / LRP1 regulates cancer cell infiltration and endothelial recruitment is mediated by CTGF (FIGS. 8D, 8E).

Example 10 CTGF mediates miRNA-dependent metastatic infiltration, endothelial recruitment and colonization In this example, the assay is used to investigate in detail whether CTGF mediates miRNA-dependent infiltration and endothelial recruitment. went. Briefly, transwell cell infiltration and endothelial recruitment assays were performed on parental MeWo cells overexpressing miR-199a or miR-1908 in the presence of blocking antibodies targeting CTGF. In fact, it was found that mir-199a and mir-1908-dependent metastatic infiltration and endothelial recruitment are mediated by CTGF (FIGS. 9A and 9B). To investigate in detail whether in vivo melanoma metastasis (metastatic colony formation) is mediated by CTGF, bioluminescence imaging was knocked down for CTGF in the presence of overexpression of miR-199a or miR-1908. It was performed for lung metastasis by 5 × 10 ⁴ parental MeWo cells. Knockdown of CTGF in this situation resulted in a significant reduction in melanoma metastasis in vivo (Fig. 9C).

Example 11 Treatment with GW3965 of the LXR agonist increases ApoE and DNAJA4 levels of melanoma cells, and various small molecule agonists of the liver X receptor (LXR) that suppress cancer cell infiltration, endothelial recruitment and metastatic colony formation It has been previously shown to increase the level of ApoE. Various assays were performed to investigate whether increasing Apo-E levels through LXR activation provided therapeutic benefits, including Apo-E levels, tumor cell infiltration, endothelial recruitment and in vivo. LXR agonist GW3965 (chemical name: 3- [N- (2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl) amino] propyloxy] phenylacetate hydrochloride for melanoma transfer in ) Was evaluated (Fig. 10). Incubation of parental MeWo cells in the presence of therapeutic concentrations of GW3965 increased the expression of ApoE and DNAJA4 (FIGS. 10A and 10B). Pretreatment of MeWO cells with GW3965 reduced tumor cell infiltration (Fig. 10C) and reduced endothelial recruitment (Fig. 10D). To test whether GW3965 can inhibit metastasis in vivo, mice were fed a grain-type solid diet or control diet containing GW3965 (20 mg / kg) and lung metastases were 4 × 10 ⁴ cells. Parental MeWo cells were injected into the tail vein into mice and then assayed using bioluminescence (FIG. 10E). Oral administration of GW3965 to mice in this manner resulted in a significant reduction in melanoma metastasis in vivo (FIG. 10E).

Example 12 Identification of miR-7 as an endogenous suppressor of melanoma metastasis In this example, miR-7 was identified as an endogenous suppressor of melanoma metastasis (FIG. 11). To test whether miR-7 suppresses melanoma metastasis in vivo, its expression was knocked down in parental MeWo cells using miR-Zip technology (FIG. 11A). Bioluminescence image of metastatic colony formation in the lung after intravenous infusion of 4 × 10 ⁴ parental MeWo cells (miR-7 KD) expressing a short hairpin (miR-Zip) inhibitor of miR-7. The bioluminescence plot significantly increased lung metastases in vivo (FIG. 11A). Conversely, overexpression of miR-7 in LM2 cells markedly reduced lung metastases in vivo (FIG. 11B).

Cancer complexity requires the application of systematic analysis (Pe'er and Hacohen, 2011). Through systematic overall efforts, collaborative networks of various miRNAs have been discovered. These miRNAs are i) upregulated in highly metastatic human melanoma cells, ii) required and sufficient for metastatic colony formation and angiogenesis in melanoma, and ii) metastatic human melanoma. It is a solid pathological predictor of sexual recurrence. Through transcriptome-based and biologically-guided targeting efforts, miR-1908, miR-199a-3p and miR-199a-5p converge on heat shock factor DNAJA4 and metabolic gene ApoE. It was found to be targeted. The requirement for each individual miRNA for metastasis indicates that these three convergent miRNAs are non-redundant in promoting melanoma transfer, while being achieved by combinatorial miRNA inhibition. Robust synergistic suppression of metastasis reveals functional cooperation between these miRNAs, which may be achieved through maximal silencing of ApoE or DNAJA4. .. Suppression of melanoma progression by identifying ApoE as a gene that is negatively regulated by the three metastasis-promoting factors miRNA, positively regulated by the metastasis-suppressing factor gene (DNAJA4), and silenced in clinical metastasis samples. The importance of this gene as a factor is emphasized.

Example 13 Identification of LXRβ signaling as a novel therapeutic target in melanoma In order to identify nuclear hormone receptors that exhibit widespread expression in melanoma, we have determined the expression levels of all nuclear hormone receptor family members. The human melanoma cell line was examined over the NCI-60 collection. Several receptors exhibited stable expression across multiple melanoma strains, suggesting that these receptors may represent novel potential targets in melanoma (FIGS. 19A and 20A). Notably, of these, the liver X receptor (LXR) has previously been shown to enhance ApoE transcription in adipocytes and macrophages (Laffitte et al., 2001), while the pharmacology of RXR. Activation was found to cause expression of ApoE in a preclinical Alzheimer's model (Cramer et al., 2012).

Given the recently discovered metastasis-suppressing role of ApoE in melanoma (Pencheva et al., 2012), ubiquitous basal expression of LXRβ and RXRα in melanoma, as well as therapeutic activation of various LXRs and RXRs. Given the availability of pharmacological agents for this, we investigated in detail whether activation of LXRs or RXRs in melanoma cells may inhibit the melanoma progression phenotype. Given the proven role of nuclear hormone receptors (eg, ER and AR) in breast cancer cell proliferation and prostate cancer cell proliferation, we first first pharmacologically agonize LXR or RXR in melanoma cells. Was investigated to affect in vitro cell growth.

Treatment of melanoma cells with two structurally distinct LXR agonists, namely GW3965 [2] or T0901317 [1], or with the RXR agonist bexarotene, did not affect cell proliferation or cell viability (percentage of cell proliferation or cell viability). 20B to 20C). We then evaluated the effects of LXR or RXR activation on cell infiltration and endothelial recruitment, which are the phenotypes exhibited by the metastatic melanoma population and the metastatic breast cancer population (Pencheva). Et al. 2012; Png et al., 2012). Mutual diversity of MeWo (B-Raf / N-Ras wild type) human melanoma line, HT-144 (B-Raf variant) human melanoma line and SK-Mel-2 (N-Ras variant) human melanoma line, Similarly, treatment with GW3965 [2] or T0901317 [1] of the SK-Mel-334.2 (B-Raf variant) primary human melanoma strain causes melanoma cells to infiltrate through Matrigel in a transwell assay. It consistently suppressed the ability to and the ability to recruit endothelial cells (FIGS. 19B-19C). In comparison, treatment with bexarotene suppressed infiltration only in half of the melanoma strains tested and had no significant effect on the endothelial mobilization phenotype (FIGS. 19B-19C).

Given that LXR agonism is superior to RXR agonism in broadly inhibiting both cell infiltration and endothelial recruitment across multiple melanoma lines, we mediate the inhibitory effects of LXR agonists. It was investigated in detail that LXR signaling is required in. Knockdown of LXRβ of melanoma, not LXRα, revoked the infiltration-suppressing and endothelial mobilization-suppressing abilities of GW3965 [2] and T0901317 [1] (FIGS. 19D-19G and 20D-20G). LXRβ in melanoma cells has been shown to be a functional target for LXR agonists in inducing suppression of these in vitro phenotypes. Our molecular findings are consistent with LXRβ being the predominant LXR isotype expressed by melanoma cells (FIG. 19A, P <0.0001).

The ubiquitous basal expression of LXRβ in melanoma probably reflects the overall role that LXR plays in controlling lipid transport, synthesis and catabolism (Calkin and Totonoz, 2013). While such stable LXRβ expression appears to be important for maintaining metabolism and growth of melanoma cells, such stable LXRβ expression also provides LXR signaling, a wide range of treatments in melanoma. It makes it an attractive candidate for targeted targeting.

Example 14 Therapeutic delivery of LXR agonists suppresses melanoma tumor growth Various LXR agonists were first developed as oral drug candidates for the purpose of lowering cholesterol in patients with dyslipidemia and atherosclerosis. (Cholesterol et al., 2002; Joseph and Tontonoz, 2003). These compounds were clinically abandoned with the inability to reduce lipid levels in preclinical models of large animals (Groot et al., 2005).

Given the robust ability of GW3965 [2] and T0901317 [1] to suppress the melanoma progression phenotype in vitro (FIGS. 19B-19C), we consider therapeutic LXR activation to treat melanoma. Investigated whether it could be used for. In fact, oral administration of GW3965 [2] or T0901317 [1] at low doses (20 mg / kg) is aggressive in immune-eligible models after the formation of subcutaneous tumors that are 5 mm ^3-10 mm ³ in volume. Tumor growth by B16F10 mouse melanoma cells was suppressed by 67% and 61%, respectively (FIGS. 21A-21B). Administration of a larger LXR agonist dose (100 mg / kg) resulted in an 80% reduction in tumor growth (FIG. 21A), consistent with a dose-dependent inhibitory effect.

Oral administration of GW3965 [2] also tightly suppressed tumor growth by the MeWo human melanoma cell line (70% inhibition) and tightly suppressed tumor growth by the SK-Mel-2 human melanoma cell line (49%). (Inhibition), as well as tightly suppressed tumor growth by the SK-Mel-334.2 primary human melanoma line (73% inhibition) (FIGS. 21C-21E and 22A).

Encouraged by the robust tumor suppressive effects of LXR agonists ^{on small tumors (5 mm 3-10 mm 3 )} (FIGS. 21A-21E), we next have a large LXR activation therapy (approximately 150 mm ³ ). ) We investigated in detail whether it could inhibit the growth of tumors.

We have found that treatment with GW3965 [2] caused a roughly 50% reduction in the growth of established large B16F10 tumors (FIG. 21F). Importantly, therapeutic delivery of GW3965 [2] after tumor establishment includes immunoeligible mice injected with mouse B16F10 cells, immunocompromised mice with tumor xenografts derived from human MeWo established melanoma strains, as well. In addition, SK-Mel. 334-2 The overall survival of immunocompromised mice with tumor xenografts from the primary human melanoma line was substantially extended (FIGS. 21G-21I). These findings are consistent with the broad-spectrum responsiveness of LXR activation therapy across black and non-pigmented established melanoma tumors of the various mutant subtypes below: wild-type B-Raf and N-Ras ( B16F10 and MeWo; FIGS. 21A-21C), B-Raf mutant (SK-Mel-334.2; FIG. 21D), N-Ras mutant (SK-Mel-2; FIG. 21E).

We then sought to clarify the cell biological phenotype regulated by LXR agonists in suppressing tumor growth. Consistent with the inhibitory effect of GW3965 [2] on endothelial recruitment by melanoma cells in vitro, administration of GW3965 [2] caused a roughly half reduction in tumor endothelial cell content. (Fig. 21J). This effect was accompanied by a modest reduction (23%) in the number of actively growing tumor cells in vivo (Fig. 21K) and no change in the number of apoptotic cells (Fig. 21L). These results show that in addition to reducing local tumor infiltration, LXR activation suppresses melanoma tumor growth primarily by inhibiting tumor angiogenesis with the resulting reduction in in vivo growth. Suggest that.

Example 15 LXR agonism suppresses melanoma metastasis to lung and brain and inhibits the progression of early metastasis. The strong inhibitory effect of LXR agonists on melanoma tumor growth is that LXR activation also suppresses LXR activation. It motivated us to investigate whether metastatic colony formation by melanoma cells could be suppressed. To this end, pretreatment of human MeWo melanoma cells with GW3965 [2] caused a> 50-fold reduction in their metastatic colonization potential (FIG. 23A). In light of this dramatic inhibitory effect, we then evaluated whether orally administered LXR agonists could suppress metastasis. In immunocompromised mice orally administered with GW3965 [2] or T0901317 [1], a 31-fold and 23-fold reduction occurred in metastatic colonization in the lung by human MeWo cells, respectively. (FIGS. 23B to 23C). Treatment with GW3965 [2] also suppressed metastatic colonization by the HT-144 melanoma line (FIG. 23D) and also suppressed metastatic colonization by the SK-Mel-334.2 primary melanoma line. (Fig. 23E).

GW3965 [2] is a lipophilic molecule capable of efficiently crossing the blood-brain barrier and strongly activating LXR signaling in the brain. Consistent with this, oral delivery of GW3965 [2] has previously been shown to improve amyloid plaque pathology and memory deficiency in a preclinical model of Alzheimer's disease (Jiang et al., 2008). Therefore, we are not able to show therapeutic activity in LXR agonism in the suppression of melanoma brain metastases, i.e., the feared melanoma outcomes (Fonchem et al., 2012) that urgently require effective treatment. I thought. Notably, oral administration of GW3965 [2] inhibited both systemic dissemination and colonization in the brain after intracardiac injection of brain metastatic melanoma cells from the MeWo parent line (Fig.). 23F). These results reveal robust metastatic inhibition by LXR activation therapy across multiple melanoma lines and at multiple distal organ metastatic sites.

Encouraged by the robust effect observed in suppressing metastasis formation (FIGS. 23A-23F), we then LXR activation therapy to stop the progression of melanoma cells already scattered by the metastasis. I tried to clarify whether it could be done. We first tested whether GW3965 [2] could reduce lung colonization by melanoma cells scattered from sympatric sites after primary tumor removal (FIG. 23G). Importantly, oral administration of GW3965 [2] after tumor resection inhibited colonization in the lung by scattered melanoma cells by a factor of 17 (FIG. 23H). Notably, treatment of mice with GW3965 [2] also dramatically (1/28) suppressed colonization by early lung metastases that were eight-fold advanced from baseline at seeding (1/28). FIG. 23I). Consistent with LXR activation that inhibits the initiation of metastasis, treatment with GW3965 [2] reduced the number of macroscopic metastatic nodules formed (FIG. 23J). Finally, treatment of mice with GW3965 [2] in this'auxiliary'preclinical situation significantly prolonged mouse survival after metastatic colonization (Fig. 23K).

Example 16 LXR activation reduces melanoma progression and metastasis in a genetically driven mouse model of melanoma Roughly 60% of human melanoma tumors are characterized by activating mutations in the Braf oncogene. One amino acid variant, namely B-Raf ^V600E , is the dominant mutation found (Davies et al., 2002). Approximately 20% of melanomas show activating mutations in B-Raf with simultaneous silencing of Pten tumor suppressors, leading to the progression to a malignant melanoma state (Tsao et al.). , 2004; Chin et al., 2006). In recent years, tyrosinase (Tyr) -driven conditional B-Raf activation and Pten loss have been shown to be genetically cooperative in driving melanoma progression in mice (Dankort et al., 2009).

To determine whether LXR activation can suppress melanoma progression in this genetic initiation model, we present melanoma by intraperitoneal administration of 4-hydroxytamoxiphen (4-HT) Tyr ::. CreER; B-Raf ^{V600E / +} ; Pten ^{lox / +} mice and Tyr :: CreER; B-Raf ^{V600E / +} ; Pten ^{lox / lox} mice. Notably, oral administration of GW3965 [2] after initiation of melanoma was performed with PTEN heterozygous Tyr :: CreER; B-Raf ^{V600E / +} ; Pten ^{lox / +} mice and PTEN homozygous Tyr :: CreER; B-Raf ^{V600E / +} ; Pten ^{lox / lox} mice both attenuated tumor progression and significantly prolonged overall survival (FIGS. 24A-24B and 25A-25B). Next, we investigated whether GW3965 [2] could suppress melanoma metastasis in this genetic context. We did not detect macroscopic metastases in the lungs or brain of 4-HT-treated Tyr :: CreER; B-Raf ^{V600E / +} ; Pten ^{lox / lox} control mice, but salivary gland lymph nodes. Consistently recognized melanoma metastasis to. Importantly, Tyr :: CreER; B-Raf ^{V600E / +} ; Pten ^{lox / lox} mice treated with GW3965 [2] showed a decrease in the number of lymphatic metastases detected after death (FIG. 24C). These findings indicate that LXR activation inhibits orthotopic metastasis in a genetically driven melanoma model, in addition to its inhibitory effect on primary melanoma tumor progression.

The cooperation between B-Raf activation and Pten loss in driving melanoma progression can be further enhanced by inactivation of CDKN2A, a cell cycle regulator that frequently mutates in familial melanoma (Hussussian et al., Et al. 1994; Kamb et al., 1994). Therefore, we investigated the effect of LXR activation on B-Raf ^{V600E / +} ; Pten ^{− / −} ; CDKN2A ^{− / −} melanoma, thereby providing the therapeutic efficacy of LXR agonism. It could be tested in a more aggressive genetically driven melanoma progression model. Importantly, therapeutic delivery of GW3965 [2] is infused into allogeneic immune-eligible mice B-Raf ^{V600E / +} ; Pten ^{− / −} ; CDKN2A ^{− / −} tumor growth and lungs by primary mouse melanoma cells. It firmly inhibited metastasis and prolonged overall survival of mice with B-Raf ^{V600E / +} ; Pten ^{− / −} ; CDKN2A ^{− / −} melanoma load (FIGS. 24D-24F). Taken together, the robust suppression of melanoma progression across xenograft and gene-induced independent immunoqualitative melanoma mouse models showing diverse mutant profiles of human melanoma motivated clinical trials of LXR activation therapy.

Example 17 Pharmacological activation of LXRβ suppresses the melanoma phenotype by inducing transcription of ApoE expression in melanoma cells. We then mediate the suppression of melanoma progression downstream of LXRβ. I tried to clarify the target. To this end, we performed transcriptome profiling of human MeWo melanoma cells treated with the LXR agonist GW3965 [2].

Of the 365 genes significantly induced in response to LXR activation, we identified ApoE, a previously identified transcription target for LXR in macrophages and adipocytes (Laffitte et al., 2001). , Identified as the most upregulated secretory factor in melanoma cells (Fig. 26). Confirmation by quantitative real-time PCR (qRT-PCR) revealed robust upregulation of ApoE transcript expression across multiple human melanoma strains after treatment with unrelated LXR agonists (FIGS. 27A-Figure). 27C).

In light of the previously reported metastasis-suppressing function of ApoE in melanoma (Pencheva et al., 2012), we investigated in detail whether LXRβ activation suppresses melanoma progression by induction of ApoE transcription. In fact, GW3965 [2] and T0901317 [1] are fused to either of the two previously characterized LXR-binding multi-enhancer elements (ME.1 or ME.2) (Laffitte et al., 2001). It was found to enhance the activity of melanoma cell drive of luciferase reporter constructs containing the ApoE promoter (FIG. 28A). Importantly, this transcriptional induction resulted in elevated levels of secreted ApoE protein (Fig. 28B). Consistent with direct LXRβ targeting of ApoE in melanoma cells, neutralization of extracellular ApoE by antibodies completely inhibits LXRβ-mediated suppression of cell infiltration and endothelial recruitment, and controls these phenotypes IgG treatment. (FIGS. 28C-28G and 27D-27F), which revealed that the effects of LXR agonism are regulated by extracellular ApoE.

In addition, molecular knockdown of ApoE in melanoma cells also blocked GW3965 [2] -mediated suppression of cell infiltration and endothelial mobilization phenotypes (FIGS. 27G-27H). Consistent with this, depletion of LXRβ, but not LXRα, in melanoma cells revoked the ability of GW3965 [2] and T0901317 [1] to upregulate ApoE transcription and ultimately protein expression. (FIGS. 28H-28I and 27I-27K). Taken together, these findings indicate that pharmacological activation of LXRβ (the dominant LXR isotype expressed by melanoma cells) transcribes cell-specific infiltration and endothelial recruitment by melanoma cells and ApoE expression in melanoma cells. It has been shown to suppress through activation.

Example 18 Involvement of melanoma-derived ApoE and systemic ApoE by LXRβ activation therapy The inhibitory effect of LXR agonists in vivo by LXRβ-induced inhibition of the important melanoma phenotype by extracellular ApoE in vitro (which is the peripheral tissue). It has been suggested that activation of LXRs in (which can serve as a robust source of extracellular ApoE) may be further enhanced.

Importantly, it is unlikely that such non-transformed tissue will be less likely to develop resistance to LXR activation therapy, which allows long-term ApoE induction in patients. Therefore, we investigated in detail whether therapeutic LXR agonism suppresses melanoma progression by inducing ApoE derived from melanoma cells or systemic tissues. Consistent with LXRβ agonism increasing ApoE expression in melanoma cells in vivo, ApoE transcript levels are also isolated in melanoma primary tumors from mice fed an LXR agonist supplement diet. Up-regulated in lung and brain metastases (FIGS. 29A-29E). Importantly, treatment of mice with either GW3965 [2] or T0901317 [1] markedly increased ApoE protein expression in mouse systemic adipocyte tissue, lung tissue and brain tissue (FIGS. 30A-Figure). 30B), and ApoE transcript levels were similarly upregulated in circulating leukocytes (FIG. 30C). These results indicate that LXR activation therapy induces ApoE expression in both melanoma cells and systemic tissues in vivo.

To demonstrate that melanoma-derived LXR activation and systemic LXR activation are required in vivo for the tumor suppressive effects of orally administered LXR agonists, we first first. It was tested whether GW3965 [2] can suppress tumor growth by B16F10 mouse melanoma cells depleted of LXRβ.

Consistent with our findings in human melanoma cells, LXRβ knockdown in mouse melanoma cells canceled GW3965-mediated induction of ApoE expression (FIGS. 29F-29H). Despite this, LXRβ knockdown of melanoma cells could not prevent the inhibition of tumor growth by GW3965 [2] (FIG. 29D), which is why systemic in inhibition of tumor growth by GW3965 [2]. A role for LXR activation has been implied. To identify the LXR isotype that mediates this tumor non-autonomous suppression of melanoma growth by LXR agonists, we present the effect of GW3965 [2] on tumors implanted in genetically null mice with LXRα or LXRβ. I checked. Interestingly, systemic LXRβ genetic loss prevented GW3965 from being able to suppress melanoma tumor growth, while inactivation of LXRα had no effect on tumor growth inhibition by GW3965. (Fig. 6D). Importantly, upregulation of systemic ApoE expression by GW3965 [2] (an agonist with 6-fold greater activity against LXRβ than LXRα) was performed in LXRβ − / − mice rather than in LXRα − / − mice. It was canceled (FIGS. 30E and 29I). These results indicate that ApoE induction by GW3965 [2] in peripheral tissues is predominantly driven by systemic LXRβ activation. Consistent with this, we have found that systemic LXRβ is the primary molecular target and effector of GW3965 [2] in mediating melanoma tumor growth inhibition.

We then investigated whether ApoE is required for the in vivo melanoma-suppressing effect of LXR agonists. Depletion of melanoma cell ApoE also did not prevent tumor growth inhibition by GW3965 [2], consistent with no effect on tumor suppressive activity of GW3965 [2] for LXRβ knockdown of melanoma cells (FIG. 29F). -FIG. 29H and FIG. 30F). These findings suggest that the tumor suppressive effects of GW3965 [2] may be mediated primarily via ApoE induction in systemic tissues.

In fact, GW3965 [2] is not completely incapacitated in suppressing tumor growth in mice genetically inactivated for ApoE (Fig. 30F), which indicates that systemic ApoE is melanoma tumor growth. It was revealed as a downstream effector of systemic LXRβ in pushing for inhibition. Interestingly, in contrast to the regulation of primary tumor growth, ApoE knockdown of melanoma cells partially blocked the metastasis-suppressing effect of GW3965 [2] (Fig. 30G). Similarly, genetic inactivation of ApoE only partially prevented GW3965 [2] -induced metastatic suppression (Fig. 30G). GW3965-led inhibition of metastasis is completely blocked only in the context of both melanoma cell ApoE knockdown and genetic inactivation of systemic ApoE (FIG. 30G), thus the involvement of melanoma-derived ApoE by LXRβ and It has been implied that both systemic ApoE involvement is required in suppressing metastasis. Thus, we present that the effect of LXRβ activation on primary tumor growth is elicited primarily by systemic ApoE induction, while the effect of LXRβ agonism on metastasis is that of ApoE in both melanoma cells and systemic tissues. We conclude that it is mediated by transcriptional induction.

The identification of ApoE as the only downstream mediator of melanoma phenotypic LXRβ-induced inhibition further underscores the importance of this gene as an inhibitor of melanoma progression. To determine whether ApoE expression provides a clinical prognosis for metastatic outcomes of melanoma, we have surgically resected 71 human primary melanoma lesions at ApoE protein levels. Was assessed by performing a blinded immunohistochemical analysis.

We find that patients with melanoma metastases showed roughly one-third reduction in ApoE expression in those primary tumors compared to patients without melanoma metastases. (FIG. 30H, P = 0.002). Notably, the level of ApoE expression in the patient's primary melanoma lesions strongly stratified patients at high risk for metastatic recurrence from those at low risk (FIGS. 30I, P). = 0.002). These observations are consistent with previous findings (Pencheva et al., 2012) in which significantly lower levels of ApoE in the distal melanoma metastases were revealed for primary lesions. In summary, the study found that ApoE is the only gene, i) at risk for metastatic recurrence of melanoma, and as such, ii) clinical benefit from LXRβ agonist-mediated ApoE induction. It has been shown that it may possibly act as a prognostic and predictive biomarker in primary melanoma to identify patients who may be able to obtain it.

Example 19 LXRβ activation therapy suppresses the growth of melanoma that is resistant to dacarbazine and vemurafenib LXRβ activation therapy robustly promotes melanoma tumor growth and metastasis across a wide range of melanoma lines with diverse mutation backgrounds Encouraged by being able to suppress, we then resist two of the leading clinical agents used in the management of metastatic melanoma, namely dacarbazine and vemurafenib. Attempts were made to determine whether sex melanoma could respond to LXRβ activation therapy.

To this end, we created B16F10 clones that are resistant to dacarbazine (DTIC) by continuously culturing melanoma cells in the presence of DTIC for 2 months. This resulted in a population of cells that showed a 7-fold increase in viability in response to high doses of DTIC treatment compared to the parental B16F10 cell line (FIG. 31A). To confirm that this in vitro-derived DTIC clone is also resistant to DTIC in vivo, we evaluated the effect of dacarbazine treatment on tumor growth.

Dacarbazine significantly suppressed the growth of DTIC-sensitive parent lines (Fig. 31B), but did not affect tumor growth by DTIC-resistant cells of B16F10 (Fig. 31C). GW3965 [2] firmly suppressed tumor growth by DTIC-resistant B16F10 melanoma clones by more than 70% (FIGS. 31C-31D). Importantly, oral delivery of GW3965 [2] also strongly inhibited the growth of in vivo-derived DTIC-resistant human melanoma tumors formed by unrelated MeWo cell lines (FIGS. 31E-31F and 32A). ).

These results indicate that LXRβ agonism is effective in suppressing a large population of melanoma cells that are resistant to dacarbazine, the only FDA-approved cytotoxic chemotherapeutic agent in metastatic melanoma. It will be revealed. Our findings have important clinical implications for the treatment of melanoma. This is because all stage IV patients treated with dacarbazine eventually progress by developing tumors that are resistant to this drug.

We have a mode of treatment that is active against the recently approved B-Raf kinase inhibitor vemurafenib, ie, against B-Raf mutant melanoma (Bollag et al., 2010; Sosman et al., 2012). ) Was tested for the effect of LXRβ activation therapy on melanoma cells that are resistant to. Numerous researchers have derived melanoma strains that are resistant to vemurafenib (Poulikakos et al., 2011; Shi et al., 2012; Das Thakur et al., 2013). Treatment with GW3965 [2]
It suppressed the growth of the previously induced vemurafenib-resistant SK-Mel-239 strain by 72% (Fig. 31G) and significantly prolonged the survival of mice loaded with vemurafenib-resistant melanoma (Fig. 31H). Based on our findings from combined pharmacological, molecular and genetic studies in various preclinical models of melanoma, LXRβ targeting apoE (melanoma) tumor growth and metastasis of melanoma. It is demonstrated as a novel therapeutic effort to firmly suppress by transcriptional induction of invasion and metastatic angiogenesis (Pencheva et al., 2012; FIG. 31I).

Example 20 Treatment with ApoE suppresses tumor cell infiltration and endothelial recruitment across a large number of cancer types, including breast, renal cell and pancreatic cancers ApoE treatment treats various cancer types in addition to melanoma In vitro assays are performed to determine if it may be effective for ApoE treatment against several different cancer cell lines, including breast cancer cell lines, renal cell cancer cell lines and pancreatic cancer cell lines. The impact was evaluated (Fig. 33).

The ability of cancer cells to infiltrate through Matrigel in vitro was tested using the Transwell Matrigel Infiltration Chamber System (354480, BD Biosciences). Various cancer cell lines were subjected to overnight serum starvation in medium containing 0.2% FBS. The next day, the infiltration chamber was pre-equilibrated prior to the assay by adding 0.5 mL of starvation medium to the top and bottom wells. In the meantime, cancer cells were trypsinized and live cells were counted using trypan blue dead cell exclusion dye. The cancer cells are then resuspended at a concentration of 1 × 10 ⁵ cells per 1 mL of starvation medium and 0.5 mL of cell suspension (containing 5 × 10 ⁴ cells) is placed in each transwell. Sown. To clarify the effect of recombinant ApoE on cancer cell infiltration, human recombinant ApoE3 (4696, Biovision) or BSA was added to each transwell at 100 μg / mL at the start of the assay. The infiltration assay was run at 37 ° C. for 24 hours. When the assay is complete, the inserts are washed with PBS and the uninfiltrated cells are gently scraped from the top surface of each insert using a q-chip and the cells infiltrated into the base surface of the insert. Was fixed at 4% PFA at room temperature for 15 minutes. After the fixation treatment, the inserts were washed with PBS and then excised and fixed to slides using a VectorSiels fixation medium containing DAPI nuclear staining (H-1000, Vector Laboratories). The base surface of each insert was imaged using an inverted fluorescence microscope (Zeiss Axiovert 40 CFL) at a magnification of 5x and the number of DAPI positive cells was quantified using ImageJ.

In fact, treatment with ApoE inhibited both tumor cell infiltration and endothelial recruitment across all three of these cancer types (FIGS. 33A-33I).

Example 21 LXR agonists LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) and SB7422881 are LXR agonists GW3965 [2] that induce ApoE expression in human melanoma cells. Given that ApoE activation by treatment with and T0901317 [1] provided therapeutic benefits for inhibiting tumor growth and metastasis, we next include other LXR agonists as ApoE. It was investigated whether expression could be induced in human melanoma cells (Fig. 34).

To clarify the effect of various LXR agonists (LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) and SB7422881) on ApoE expression in melanoma cells 1x 105 human ^MeWo melanoma cells were seeded in 6-well plates. The next day, DMSO or each LXR agonist was added to the cell medium at a concentration of 500 nM, 1 μM or 2 μM as shown and the cells were incubated at 37 ° C. for 48 hours in the presence of DMSO or drug. The total amount of DMSO added to the cell medium was kept below 0.2%. RNA was extracted from whole cell lysate using Total RNA Purification Kit (17200, Norgen). For each sample, 600 ng of RNA was reverse transcribed into cDNA using the cDNA First-Strand Synthesis Kit (Invitrogen). Approximately 200 ng of cDNA was mixed with SYBR® Green PCR Master Mix and the corresponding forward and reverse primers that are specific for detecting human ApoE. Each reaction was performed in quadruples and ApoE mRNA expression levels were measured by quantitative real-time PCR amplification using the ABI Prism 7900HT Real-Time PCR System (Applied Biosystems). Relative ApoE expression was determined using the ΔΔCt method. GAPDH was used as an endogenous control for normalization purposes.

In fact, treatment with the above LXR agonists (LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) and SB7422881) all caused varying degrees of induction of ApoE expression. (FIGS. 34A to 34C).

Example 22 Treatment with GW3965 of LXR agonist inhibits in vitro tumor cell infiltration of kidney cancer, pancreatic cancer and lung cancer We found that treatment with LXR agonist resulted in inhibition of melanoma tumor cell infiltration. Has been clarified. Given that this effect is mediated by activation of ApoE expression, we believe that treatment with LXR agonists will result in inhibition of in vitro tumor cell infiltration in breast, pancreatic and kidney cancer. Assumed. This is because these cancer types were responsive to ApoE treatment. To test this hypothesis, we performed an in vitro tumor cell infiltration assay by treating breast cancer cell lines, pancreatic cancer cell lines and renal cell cancer cell lines with the LXR agonist GW3965 [2]. (Fig. 35).

Various cell lines (5x10 ⁴ RCC human renal cancer cells, 5x10 ⁴ PANC1 human pancreatic cancer cells and 5x10 ⁴ H460 human lung cancer cells) were generated by DMSO or GW3965 at 1 μM 56. Time processed. Cells were subjected to 16 hours of serum starvation in medium with 0.2% FBS in the presence of DMSO or GW3965. After serum starvation, cells were subjected to a transwell infiltration assay using the Matrigel infiltration chamber system (354480, BD Biosciences). The infiltration chamber was pre-equilibrated prior to the assay by adding 0.5 mL of starvation medium to the top and bottom wells. In the meantime, cancer cells were trypsinized and live cells were counted using trypan blue. The cancer cells are then resuspended at a concentration of 1 × 10 ⁵ cells per 1 mL of starvation medium and 0.5 mL of cell suspension (containing 5 × 10 ⁴ cells) is placed in each transwell. Sown. The infiltration assay was run at 37 ° C. for 24 hours. When the assay is complete, the inserts are washed with PBS and the uninfiltrated cells are gently scraped from the top surface of each insert using a q-chip and the cells infiltrated into the base surface of the insert. Was fixed at 4% PFA at room temperature for 15 minutes. After the fixation treatment, the inserts were washed with PBS and then excised and fixed to slides using a VectorSchild fixing medium containing DAPI nuclear staining (H-1000, Vector Laboratories). The base surface of each insert was imaged using an inverted fluorescence microscope (Zeiss Axiovert 40 CFL) at a magnification of 5x and the number of DAPI positive cells was quantified using ImageJ.

In fact, treatment with GW3965 [2] resulted in inhibition of tumor cell infiltration in all three cancer types tested (FIGS. 35A-35C). This further demonstrated the wide therapeutic potential of LXR agonists for treating various cancer types.

Example 23 Treatment with GW3965 of LXR agonist inhibits tumor growth of breast cancer in vivo We show that LXR agonist inhibits in vitro cancer progression phenotype in breast, pancreatic and kidney cancer. I have to. To investigate in detail whether treatment with an LXR agonist inhibits primary tumor growth of breast cancer in vivo, mice injected with MDA-468 human breast cancer cells are supplemented with a control diet or LXR agonist GW3965 [2]. Treated with either of the following baits (Fig. 36).

To clarify the effect of orally delivered GW3965 [2] on breast cancer tumor growth, 2 × 10 ⁶ MDA-468 human breast cancer cells were resuspended in 50 μL PBS and 50 μL Matrigel and cell suspension. Was injected into the lower memory fat pad of both 7-week-old Nod Scid gamma female mice. Mice were assigned to control diet treatment or GW3965 supplemental diet treatment (75 mg / kg / day) 2 days prior to cancer cell infusion. The drug compound of GW3965 [2] is described in Research Diets, Inc. Was added to the mouse solid feed. Tumor size was measured using a digital caliper and tumor volume was calculated as (minimum diameter) ² x (maximum diameter) / 2.

Treatment with GW3965 resulted in a significant reduction in breast cancer tumor size in vivo (FIG. 36).

Example 24 Effects of treatment with LXR agonists (LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) and SB7422881) for melanoma progression phenotype in vitro. Reveals that various LXR agonists can induce ApoE expression with different potency in melanoma cells (FIG. 34). Since the therapeutic effect of an LXR agonist on cancer is through activation of ApoE expression, we have found that the therapeutic efficacy of a given LXR agonist of any LXR agonist is directly related to its ability to induce ApoE expression. It was assumed that it could be correlated with. To confirm this, we quantified the effects of treatment with various LXR agonists on in vitro endothelial recruitment and tumor cell infiltration of melanoma cells. As shown in FIG. 37, the degree to which the LXR agonist inhibits the cancer progression phenotype in vitro is associated with the ApoE-inducing efficacy of the LXR agonist.

Cell Infiltration: MeWo human melanoma cells were treated with DMSO, LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) or SB7422881 at 1 μM for 56 hours each. The cells were then subjected to 16 hours of serum starvation in medium with 0.2% FBS in the presence of the respective corresponding drug or DMSO. After serum starvation, cells were subjected to a transwell infiltration assay using the Matrigel infiltration chamber system (354480, BD Biosciences). The infiltration chamber was pre-equilibrated prior to the assay by adding 0.5 mL of starvation medium to the top and bottom wells. In the meantime, cancer cells were trypsinized and live cells were counted using trypan blue. The cancer cells are then resuspended at a concentration of 2 × 10 ⁵ cells per 1 mL of starvation medium and 0.5 mL of cell suspension (containing 1 × 10 ⁵ cells) is placed in each transwell. Sown. The infiltration assay was run at 37 ° C. for 24 hours. When the assay is complete, the inserts are washed with PBS and the uninfiltrated cells are gently scraped from the top surface of each insert using a q-chip and the cells infiltrated into the base surface of the insert. Was fixed at 4% PFA at room temperature for 15 minutes. After immobilization, inserts were washed with PBS, excised, and fixed to slides using VectorSheels immobilization medium containing DAPI nuclear staining (H-1000, Vector Laboratories). The base surface of each insert was imaged using an inverted fluorescence microscope (Zeiss Axiovert 40 CFL) at a magnification of 5x and the number of DAPI positive cells was quantified using ImageJ.

Endothelial recruitment: MeWo human melanoma cells were treated with DMSO, LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) or SB7422881 at 1 μM for 56 hours each. Subsequently, 5 × 10 ⁴ cancer cells were seeded in 24-well plates in the presence of each drug or DMSO and adhered for 16 hours prior to initiation of the assay. Human umbilical vein endothelial cells (HUVEC cells) were subjected to serum starvation overnight in medium containing 0.2% FBS. The next day, 1 × 10 ⁵ HUVEC cells were seeded into a 3.0 μm HTS Fluroblock insert (351151, BD Falcon) fitted into each well containing cancer cells at the bottom. HUVEC cells were allowed to migrate towards cancer cells for 20 hours, after which the inserts were washed with PBS, fixed with 4% PFA, labeled with DAPI and fixed to slides. The base surface of each insert was imaged using an inverted fluorescence microscope (Zeiss Axiovert 40 CFL) at a magnification of 5x and the number of DAPI positive cells was quantified using ImageJ.

LXR agonists that strongly induce ApoE expression (eg, WO-2010-0138598 (Ex. 9) and SB7422881) are more effective than the less potent LXR agonists in inhibiting the cancer progression phenotype (eg, WO-2010-0138598 (Ex. 9) and SB7422881). FIG. 37). This further demonstrates that the therapeutic benefit of LXR agonist treatment is the result of ApoE induction.

Example 25 Treatment with LXR agonists inhibits melanoma tumor growth in vivo We have shown that various LXR agonists that induce ApoE expression inhibit in vitro tumor activity. To determine if these agonists inhibit melanoma tumor growth in vivo, mice injected with B16F10 melanoma cells were injected with LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex. 19). It was treated with either Ex.9) or SB7422881.

5 × 10 ⁴ B16F10s to assess the effect of orally administered LXR-623, WO-2007-002563 (Ex.19), WO-2010-0138598 (Ex.9) or SB7422881 on melanoma tumor growth. Mouse melanoma cells were resuspended in 50 μL PBA and 50 μL Matrigel, and cell suspensions were subcutaneously injected into the lower dorsal flanks on both sides of 7-week-old C57BL / 6 mice. Mice were palpated daily for tumorigenesis and after tumors measuring 5 m ³ to 10 m ³ in volume were detected, mice were assigned to a control solid diet or a solid diet containing each of the following LXR agonists: LXR. -623 (20 mg / kg / day), WO-2007-002563 (Ex.19) (100 mg / kg / day), WO-2010-0138598 (Ex.9) (10 mg / kg / day or 100 mg / kg / day) ) Or SB742881 (100 mg / kg / day). These LXR agonist drug compounds are described in Research Diets, Inc. Was added to the mouse solid feed. Tumor size was measured using a digital caliper and tumor volume was calculated as (minimum diameter) ² x (maximum diameter) / 2.

Consistent with our in vitro data, LXR agonists (WO-2010-0138598 (Ex. 9) and SB7422881) that strongly induce ApoE expression in vitro significantly inhibit melanoma primary tumor growth in vivo. (Fig. 38). This also demonstrates that other LXR agonists (GW3965 [2], T0901317 [1]) that potently induce ApoE expression also inhibit primary tumor growth in vivo (the results of our inventors. This is consistent with FIG. 21).

Therefore, the above examples have focused on characterizing the molecular and cellular mechanisms that exert their effects. To this end, it has been found that ApoE targets two different, nevertheless homologous receptors in two different cell types. ApoE, which acts on the LRP1 receptor of melanoma cells, inhibits melanoma infiltration, while its action on the LRP8 receptor of endothelial cells suppresses endothelial migration. Results from loss-of-function analysis, function acquisition analysis, epistasis analysis, clinical correlation analysis and in vivo selective derivative expression analysis show that three miRNAs convergently target the metastatic inhibitor network to limit ApoE secretion. Therefore, a model that suppresses ApoE / LRP1 signaling to melanoma cells and ApoE / LRP8 signaling to endothelial cells is obtained (Fig. 7K). Although ApoE and DNAJA4 have been identified as key targets and direct mediators of the metastatic phenotype regulated by these miRNAs by the above systematic analysis, these three miRNAs are silencing. It cannot be ruled out that may individually carry additional genetic targets that may contribute to metastatic progression. However, the ability of ApoE or DNAJA4 knockdown to completely restore the metastasis-suppressing phenotype observed with individual miRNA silencing indicates that these genes are important mediators of miRNA-dependent effects on metastasis. Strongly suggest.

The above results provide combined molecular, genetic and in vivo evidence for a required and sufficient role for ApoE in the suppression of melanoma metastatic progression. ApoE can be distributed in the circulatory system in both lipoprotein-bound and lipid-free states (Hatters et al., 2006). Although lipid-free recombinant ApoE has been shown to be sufficient to suppress melanoma infiltration and endothelial migration, ApoE contained in lipoprotein particles can also suppress melanoma infiltration and endothelial recruitment. It is possible that Recombinant ApoE can inhibit these metastasis-supporting phenotypes, as well as the increased melanoma infiltration and endothelial recruitment phenotypes observed with antibody-mediated ApoE neutralization, that the ApoE molecule itself has these expressions. It suggests that it is an important mediator of the type. Consistent with the findings disclosed herein, synthetic peptide fragments of ApoE were previously found to inhibit endothelial migration via an unknown mechanism (Bhattacharjee et al., 2011). The findings disclosed herein are consistent with the role of melanoma cell secretion for ApoE and systemic endogenous ApoE in inhibiting endothelial recruitment, in which case this role is impaired endothelium. It is not secondary to cell growth.

The above molecular, genetic and in vivo studies reveal a role for endogenous cancer-derived ApoE in the regulation of endothelial migration and cancer angiogenesis through signaling of the endothelial LRP8 receptor. This robust, cell-non-autonomous endothelial recruitment phenotype mediated by ApoE / LRP8 signaling suggests that ApoE may also regulate metastatic angioplasty in other cancer types. And, such an overall role for ApoE in cancer angiogenesis must be considered in the future. ApoE is a polymorphic molecule with a well-established role in lipid disorders, cardiovascular disorders and neurodegenerative disorders. The three major variants, namely ApoE2, ApoE3 and ApoE4, show various presentations in the human population, with ApoE3 being the most common variant (Hatters et al., 2006). These three isotypes are Apo
It differs at the residues at positions 112 and 158 in the N-terminal domain containing the E-receptor binding domain. It is believed that these structural changes give rise to different functional attributes among the mutants. Consistent with this, these three ApoE isotypes differ in their binding affinity for members of the LDL receptor family, lipoprotein binding preferences, and N-terminal stability. That is, ApoE2 has a weaker LDL receptor binding ability of 1/50 to 1/100 of that of ApoE3 and ApoE4 (Weisgraver et al., 1982), while ApoE4 has two other variants. Unlike, it preferentially binds to larger, lower density lipoproteins (Weisgraver et al., 1990) and also exhibits the lowest N-terminal stability (Morrow et al., 2000). These functional differences result in pathophysiological properties for selecting the isotype of ApoE. ApoE3 (which is found in 78% of the population) is considered to be a neutral allele, but ApoE2 is associated with type III hyperlipoproteinemia (Hatters et al., 2006) and ApoE4 is for Alzheimer's disease. It represents a major known genetic risk (Corder et al., 1993) and correlates with a modest increase in the risk of developing cardiovascular disease (Luc et al., 1994). Given that the numerous human melanoma cell lines analyzed in the above studies are homozygous for the ApoE3 allele, as well as that recombinant AoE3 can inhibit melanoma infiltration and endothelial recruitment, the above findings are , ApoE3 is sufficient and consistent with the requirement for suppression of melanoma metastatic progression. However, whether ApoE2 and ApoE4 can regulate these metastatic phenotypes to the same extent as ApoE3, and whether certain ApoE genotypes pose an increased risk of melanoma metastatic progression. Clarification will be the focus of attention in the future.

With the exception of surgical resection of the primary melanoma lesion, there is currently no effective treatment to prevent melanoma metastasis, and interferon therapy has a 5-year overall survival rate of only 3 based on a meta-analysis. While increasing by%, phase III clinical data demonstration of significant survival benefits remains significant (Garbe et al., 2011). The dramatic increase in melanoma metastatic progression in the context of systemic ApoE genetic loss is that regulating ApoE levels can lead to melanoma, a disease that kills approximately 48,000 people worldwide each year (Lucas et al.). , 2006) suggests that it may have important therapeutic implications. Given that ApoE can robustly suppress melanoma infiltration, endothelial migration, metastatic angiogenesis and metastatic colony formation, therapeutic efforts aimed at pharmacological induction of endogenous ApoE levels are melanoma mortality. May be significantly reduced by reducing the occurrence of metastases.

The above, unpredictable, in vivo selection-based approach is a deregulated miRNA that synergistically and dramatically promotes cancer cell metastasis from independent patient melanoma cell lines representing both black and non-pigmented melanoma. Brought about the discovery of. Although miR-1908 has not been previously characterized, hepatocellular carcinoma (Hou et al., 2011; Shen et al., 2010) and osteosarcoma in which miR-199a exhibits downregulation of miR-199a expression levels contrary to melanoma. (Duan et al., 2011). These differences are consistent with the proven tissue-specific expression profiles of miRNAs in various cancer types. The identification of miR-199a as a promoter of melanoma metastasis is supported by a previous joint clinical study (Worley et al., 2008) revealing that increased miR-199a levels correlate with the progression of uveal melanoma. This suggests that induced miR-199a expression may be a defining feature of metastatic melanoma, regardless of the site of origin. In previous studies, additional miRNAs have been associated with promoting melanoma metastatic progression: for example, miR-182 (Segra et al., 2009), miR-214 (which is upregulated in metastatic melanoma cells). However, it did not function operably in the above studies; Penna et al., 2011), and miR-30b / miR-30d (Gaziel-Sovran et al., 2011) and the like. Each of these miRNAs moderately regulates melanoma metastasis, which can lead to increased or decreased metastatic regulation by 1.5- to 2-fold differences during miRNA overexpression or knockdown, respectively. It has been reported. In contrast, overexpression of either miR-199a or miR-1908 increased metastasis 9-fold (Fig. 1C), while combinatorial miRNA knockdown synergistically suppressed melanoma metastasis by more than 70-fold difference. (Fig. 7E). Therefore, the studies disclosed herein are the first systematic discoveries of numerous miRNAs that converge and robustly promote human melanoma metastasis, as well as dual cell-autonomous / cell-non-autonomous. It represents the first to map a role to the endogenous metastasis-regulating miRNA in cancer.

Previous systematic analyzes of miRNAs in breast cancer primarily revealed reduced levels of expression of large numbers of microRNAs in in vivo selected metastatic breast cancer cells (Tavazoie et al., 2008). These findings include the subsequent discovery of many further metastasis-suppressing factor miRNAs in breast cancer (Shi et al., 2010; Wang and Wang, 2011), and the identification of several miRNAs as direct transcription targets for p53 tumor suppressors (He). Et al. 2007), MiRNA downregulation in breast cancer against normal tissues (Calin and Groce, 2006; Iorio et al., 2005), Drawsha and Dicer breast cancer (Yan et al., 2011) and metastatic breast cancer (Griller et al., 2011). ), As well as the tumorigenic and metastasis-supporting effects of general miRNA silencing mediated by dicer knockdown (Kumar et al., 2007; Kumar et al., 2009; Martello et al., 2010; Noh et al. , 2011). In contrast to breast cancer, the above findings in melanoma reveal a set of miRNAs that are upregulated in metastatic human melanoma, which means that the processing of miRNAs actually supports tumorigenesis in melanoma or It offers an interesting possibility that it may be acting in a metastasis-supporting manner. Consistent with this, dicers are required for the development of melanocytes (Levy et al., 2010), and dicer expression has recently been positively correlated with human melanoma progression in clinical pathological studies. Was found (Ma et al., 2011). These findings, when integrated with the findings disclosed herein, motivate further research to investigate further research is needed for dicers to be operable in melanoma metastasis (Bernstein et al., 2001).

The establishment of in vivo selection models of melanoma and non-pigmented melanoma metastases has made it possible to identify the cell phenotypes exhibited by hypermetastatic melanoma cells. This study reveals that, in addition to increased infiltration, the ability of melanoma cells to mobilize endothelial cells is significantly enhanced in high metastatic melanoma cells as opposed to low metastatic melanoma cells. In addition, three major post-transcriptional regulators of metastasis were found to strongly mediate endothelial recruitment. Downstream signaling pathways regulated by these miRNAs have also been found to further regulate endothelial recruitment. These findings reveal that endothelial recruitment is a defining feature of metastatic melanoma cells. Increased endothelial mobilization capacity has also recently been found to be a defining feature of metastatic breast cancer, in which case inhibition of metastasis by miR-126 promotes endothelial mobilization in two different signaling pathways. Mediated via miRNA targeting (Png et al., 2012). In breast cancer, endothelial recruitment increased the likelihood of metastasis initiation rather than tumor growth. Similarly, the melanoma metastasis-promoting factor miRNAs studied in this case dramatically increase metastatic colony formation without increasing primary tumor growth and also increase the number of metastatic nodules, which are tumors. Rather than promoting growth, it was consistent with the role of these miRNAs and their regulatory networks in metastasis initiation. Taken together, these findings are consistent with endothelial recruitment into metastatic niches that act as promoters of metastatic initiation and metastatic colonization in these different epithelial cancer types. Such a non-canonical role for endothelial cells in cancer progression would contrast with the proven role of endothelial cells in enhancing angiogenesis of bloodstreams that accelerate increased tumor growth. Endothelial cells are known to play such a non-canonical role in development by giving various cues to nearby cells during organogenesis (Lammert et al., 2001). Such cues have also been shown in recent years to promote organ regeneration (Ding et al., 2011; Ding et al., 2010; Kobayashi et al., 2010). Future studies will be needed to clarify the metastatic stimulators provided by the endothelial cells that catalyze the initiation of metastasis.

The ability to individually predict metastasis-free survival in a cohort of melanoma patients with miR-199a-3p, miR-199a-5p and miR-1908 is the respective miRNA as a clinical predictor of melanoma cancer progression. Shows the importance of. Importantly, the dramatic and critical overall signature of these three miRNAs to stratify patients at high risk for metastatic recurrence from those with essentially no risk (Figure 7D). Due to their significant ability to cooperate with these miRNAs, as well as their clinical potential as biomarkers of melanoma to identify a subset of patients who may benefit from miRNA inhibition therapy (Sawyers, 2008). ) Are revealed together. Therapeutic miRNA targeting has been shown to antagonize miRNAs in mice (Elmer et al., 2008 (b); Krutzfeldt et al., 2005; Obad et al., 2011) and primates (Elmer et al., 2008 (a)). Also, the use of in vivo LNA, which is currently being tested in human clinical trials, is gaining momentum. Strong prognostic potential of these three miRNAs, a robust synergy achieved by treating highly metastatic melanoma cells with a cocktail of LNAs targeting miR-199a-3p, miR-199a-5p and miR-1908. Demonstration by demonstrating the principle of metastasis prevention (Fig. 7E), as well as the metastasis-suppressing effect of therapeutically delivered in vivo-optimized LNAs targeting these miRNAs (Fig. 7J), for melanoma metastasis. That is, to clarify the therapeutic potential of targeting these metastasis-supporting miRNAs and angiogenesis-supporting miRNAs in combination in patients at high risk for outcomes that currently lack effective chemotherapeutic options. It motivates future clinical research aimed at.

The above examples and descriptions of preferred embodiments should be construed as exemplary rather than as limiting the invention as defined by claim. As will be readily appreciated, numerous variations and combinations of the features shown above can be utilized without departing from the invention as set forth in the claims. Such changes are not considered to deviate from the scope of the invention and all such changes are intended to be included in the claims below. All references cited herein are incorporated in their entirety.

Claims (15)

A pharmaceutical composition for treating cancer,
Contains LXRβ agonists and / or medically acceptable salts thereof,
Used in combination with nivolumab or ipilimumab ,
The LXRβ agonist was selected from the group consisting of compound 1, compound 2, compound 12 and compound 25.
A pharmaceutical composition, wherein the cancer is ovarian cancer, breast cancer, lung cancer, gliuloblastoma, melanoma, bladder cancer, renal cell cancer, colorectal cancer, lymphoma, leukemia, multiple myeloma, or hepatocellular carcinoma. ..

The pharmaceutical composition according to claim 1, wherein the cancer is ovarian cancer, breast cancer, lung cancer, glioblastoma, or melanoma. The pharmaceutical composition according to claim 1, wherein the cancer is ovarian cancer. The pharmaceutical composition according to claim 3, wherein the ovarian cancer is resistant to a platinum agent. The pharmaceutical composition according to claim 1, wherein the cancer is breast cancer. The pharmaceutical composition according to claim 5, wherein the breast cancer is a triple negative breast cancer. The pharmaceutical composition according to claim 1, wherein the cancer is lung cancer. The pharmaceutical composition according to claim 7, wherein the lung cancer is non-small cell lung cancer. The pharmaceutical composition according to claim 1, wherein the cancer is glioblastoma of the nerve. The pharmaceutical composition according to claim 1, wherein the cancer is melanoma. The pharmaceutical composition according to claim 1, wherein the LXRβ agonist is the compound 25. The pharmaceutical composition according to claim 1, wherein the cancer is metastatic. 1. Pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein the cancer has progressed during or after treatment with nivolumab or ipilimumab . The pharmaceutical composition according to claim 1, wherein the cancer is diagnosed or predicted to have resistance to nivolumab or ipilimumab .

Images